JP2013539748A - Bioactive composition comprising fig serous fraction and method for reducing the hyperpigmented appearance of skin - Google Patents

Abstract

The present invention provides a bioactive composition comprising a fig serum fraction obtained from the fig cell juice of fresh fig leaves, and a process for preparing the fig serum fraction. The composition exhibits a biological activity that can reduce skin hyperpigmentation.
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Description

This application claims the benefit of priority of US Provisional Patent Application No. 61 / 381,442, filed Sep. 10, 2010, the disclosure of which is hereby incorporated by reference in its entirety. It has been incorporated.

  The present invention relates to the field of skin whitening by topical application of a cosmetic composition to the skin. The invention further relates to a topical composition for whitening the skin, comprising a fig serum fraction. The present invention also relates to a method for reducing the appearance of hyperpigmentation of the skin by topically applying a cosmetic composition for interfering with one or more steps in melanin synthesis to the hyperpigmented area. Also related.

  Human skin includes three main layers: the epidermis, the dermis and the subcutaneous fat layer. The epidermis includes four layers: the stratum corneum (from top to bottom), the granular layer, the spiny layer and the basal layer. A separate fifth layer, a transparent layer, can be present between the stratum corneum and the granular layer. The basal layer gradually migrates upward to produce cells that form other epithelial layers. As these cells migrate upward, they lose their central nucleus and begin to produce skin proteins (keratins) and fats (lipids). These cells are identified as keratinocytes when present in the upper layers of the epidermis. Melanocytes are another class of cells located in the basal layer of the epidermis. Melanocytes are involved in the production of melanin, which is a major factor in skin pigmentation.

  Melanin is produced by a complex set of reactions within melanocytes, and at a basic level involves the enzyme tyrosinase and L-tyrosine as a substrate. Tyrosinase catalyzes the conversion of L-tyrosine to DOPA (L-3,4-dihydroxyphenylalanine) and DOPA to dopaquinone. Dopaquinone undergoes further conversion to form melanin. Melanin aggregates in organelles known as melanosomes, which are transferred to keratinocytes along elongated filaments of melanocytes known as dendrites. Approximately 1500 gene products are expressed in melanosomes, 600 of which are always expressed, and 100 of them are thought to be unique to melanosomes. In addition, there are many regulatory elements involved in signal transduction, melanosome transport within melanocytes, and movement of melanosomes into keratinocytes.

  Melanin production can be triggered by a variety of external and internal events. For example, when the skin is exposed to UV radiation, melanocytes produce additional melanin. Melanin is then transported via melanosomes to keratinocytes, which then leave a “sunburned” appearance on the skin. When UV light is removed, the melanocytes return to normal levels of melanin production. Inflammation can initiate hyperpigmentation by direct stimulation of melanocytes by mediators such as IL-1, endothelin-1 and / or stem cell factor. Reactive oxygen species generated in damaged skin or released as by-products from inflammatory cells, such as superoxide and nitric oxide, can be stimulators of melanocytes.

  Over time, chronic UV exposure, as well as other endogenous and exogenous aging factors, can cause permanent gene expression changes in keratinocytes and / or melanocytes, resulting in age-related Sexual hyperpigmentation spots occur. It has been reported that mRNA levels of some melanogenesis-related genes (eg tyrosinase, TYRP1) increase solar shoots (aging stains). The role of epithelial endothelin cascade and stem cell factor may also be emphasized in hyperpigmentation. These changes can result in invasion, eg, melanin overproduction that persists even when UV exposure is avoided and the resulting hyperpigmentation spots. In addition to hyperpigmentation stains, chronic UV exposure and other endogenous and exogenous aging factors can cause more subtle changes in skin tone. Often these changes are described as a mottled or mottled appearance. At least one study suggests that due to age-related stains, people can sometimes be seen as 10-12 years old, and the distribution of melanin may be the basis for color-dependent age decisions. ing. Accordingly, it is desirable to provide a composition and treatment method that can improve the appearance of hyperpigmented skin, for example, age-related spots.

  In recent years, consumers have increasingly demanded “natural” cosmetic products. As a result, cosmetic manufacturers have incorporated more plant-based materials into their cosmetic formulations. Various plants have been used for a variety of well-known indications for hundreds or thousands of years, but only recently have clinical confirmation of their intended effectiveness or It became possible to identify new potential uses based on the science underlying plant bioactivity. With recent advances in science, researchers can now better evaluate the effectiveness and / or potential new uses for plants that until recently were only supported by folklore. it can. Due to the novelty of this science and the extremely large number of plants that could potentially be utilized as cosmetic bioactive substances, an overwhelming number of plants have not yet been fully studied.

  Many of the methods used to extract botanical components from plants involve techniques that are detrimental to the composition of the plant tissue and / or the desired bioactive component contained in the tissue. To do. Thus, conventional extraction methods often fail to extract the full spectrum of activity present in plant cells, and therefore do not realize the full potential of botany-based cosmetic formulations. Furthermore, many conventional extraction methods utilize harsh chemical solvents, which are not “natural” and are therefore materials that consumers desire to avoid application to the skin. Furthermore, these solvent-based processes produce toxic chemical waste that can harm the environment if not properly handled and disposed of as hazardous waste.

  However, just because a material is “natural” does not mean that it does not contain undesired substances, which makes the material suitable for use on the skin. For example, many plants contain photosensitive substances, such as pheophorbide, and / or contact allergens, such as proteins. At levels found naturally in many common plants, pheophorbide and / or protein are not a concern for most people. However, when plant materials are condensed into highly concentrated forms, for example by extraction, these materials may be present at levels that cause allergic reactions, including skin irritation and rashes. However, even when these materials are present at their natural level, there are still many susceptible individuals who experience undesirable skin reactions.

  In addition, as demand for natural products increases, so does the concern about protecting the earth's natural resources. Many of the “natural” ingredients consumers desire are derived from biological resources that are depleted and / or destroyed when harvested for use in consumer products. Thus, the irony that consumers want natural, more earth-friendly products can lead to the destruction of the biological resources themselves that consumers want to preserve.

  Thus, a natural, bioactive botanical composition that maintains the desired spectrum of bioactivity, is suitable for topical application to the skin, and is prepared without the use of harsh chemical solvents There is a need for a composition. There is also a need for cosmetic compositions containing such bioactive substances that are effective in reducing areas of hyperpigmentation of the skin. There is a further need for such bioactive materials that can be harvested and processed in an ecologically reasonable and sustainable manner.

  These and other objects of the present invention will become apparent in light of the following disclosure.

  The present invention provides a fig serous fraction obtained from fresh fig leaf cell juice. The present invention also provides a cosmetic composition comprising the fig serum fraction. The fig serum fraction is present in the composition in an amount effective to achieve the desired result of whitening the skin.

  The present invention also relates to a method for reducing the appearance of hyperpigmentation of the skin by topically applying a cosmetic composition for interfering with one or more steps in melanogenesis to the hyperpigmented area. Also related.

  The present invention will be better understood from the following description when used in conjunction with the accompanying drawings. The drawings to be referred to should not be construed to limit the scope of the present invention.

FIG. 1 is a schematic diagram illustrating a process for preparing a serous fraction exhibiting biological activity obtained from fresh fig leaves. LC / UV chromatogram of a conventional fig extract superimposed on the LC / UV chromatogram of the fig serum fraction, with the conventional extract being at a higher level not detected in the fig serum fraction. It shows that it contains a slower eluting (more hydrophobic) compound. LC / UV chromatograms of slow-elution compounds of conventional fig extracts and their corresponding extracted ion chromatograms, identifying them as pheophorbide, a pigment compound that is a degradation product of chlorophyll. LC / UV chromatogram of a conventional fig extract superimposed on the LC / UV chromatogram of the fig serum fraction, indicating that the conventional extract contains higher levels of flavonol glycosides. ing. A portion of the LC / UV chromatogram of a conventional fig extract superimposed on the chromatogram of the fig serum fraction and the corresponding extracted ion chromatogram, wherein the fig serum fraction is contained in the conventional fig extract Shows approximately 10 times (10 ×) levels of catechin and related condensed tannins compared to levels contained in A portion of the LC / UV chromatogram of a conventional fig extract superimposed on the chromatogram of the fig serum fraction and the corresponding extracted ion chromatogram, the levels of the three caffeoylquinic acid isomers being It shows that it did not appear to differ substantially between the two samples. Part of the LC / MS chromatogram of a conventional fig extract superimposed on the LC / MS chromatogram of the fig serum fraction, the levels of free tyrosine, phenylalanine and tryptophan in the fig serum fraction. It shows higher. Two color photographs obtained from accelerated degradation studies showing the color stability of dried leaf fig extract compared to FSF. Two different cosmetic compositions contain different levels of fig serum fraction and different preservatives. FIG. 9 is a calibration plot for the accelerated degradation study of FIG. Color photographs obtained from accelerated aging studies comparing vehicles containing 0.55% FSF and various concentrations of preservatives.

  Unless otherwise specified, all percentages and ratios used herein relate to the weight of the entire composition and all measurements are made at 25 ° C.

  The compositions of the present invention comprise essential constituents and optional ingredients as described herein, consist essentially of essential constituents and optional ingredients as described herein, or It can consist of the essential components and optional components described in the specification. As used herein, “consisting essentially of” means that the composition or component may contain additional components, but the additional components are fundamental and novel features of the claimed composition or method. It means that it is limited to the case where it is not substantially changed.

  The term “apply” or “application”, when used with respect to a composition, means that the composition of the present invention is applied or applied onto the surface of human skin, eg, the epidermis.

  The term “dermatologically acceptable” as used herein is suitable for use in contact with human skin tissue and the composition or component described is excessively toxic, incompatible. It means that no sex, instability, allergic response, etc. occur.

  The term “safe and effective amount” as used herein means an amount of a compound or composition sufficient to significantly elicit a beneficial benefit.

  The term “hyperpigmentation after inflammation” as used herein refers to an acute to chronic increase in pigmentation as a response to a transient inflammatory event. Hyperpigmentation after inflammation is particularly prevalent in but not limited to subjects with dark skin. Hyperpigmentation after inflammation typically weakens as transient inflammatory events resolve. Examples of transient inflammatory events include, but are not limited to, pressure ulcer lesions, hair grown therein, scratches, insect bites, surface active agent damage, and short-term UV exposure.

  The term “hyperpigmented stain” as used herein is a defined area of the skin where pigmentation results from local and chronic or systemic melanin overproduction. Refers to an area that is more than the pigmentation of the skin in the adjacent area. Hyperpigmented spots are typically between about 2 mm and about 10 mm in diameter, although there may be smaller or larger spots. Hyperpigmented stains can include one or more of age-induced stains, sunlight stains, sun kuroko, hypo-melanotic lesions, freckles, and melanosis stains.

  The term “aging stain” as used herein is hyperpigmentation due to local and chronic overproduction of melanin, where pigmentation is caused by endogenous or exogenous aging factors. Refers to sexual spots.

  The term “skin tone agent” as used herein refers to melanin production signal, melanin synthesis, systemic movement of melanin between melanocytes and keratinocytes, and / or degradation of melanin. Refers to an agent that controls Skin tone agents can improve the appearance of mottled skin tone by acting as a whitening or pigmentation reducing cosmetic agent.

The term “skin tone” as used herein refers to the overall appearance of melanin in the skin caused by the synthesis of melanin, which is systemic, rather than transient. Skin tone is typically characterized over a larger area of the skin. The area may ideally be greater than 100 mm ² , but larger areas are envisaged, for example either the entire facial skin or the facial skin surface. Skin tone can be measured by image analysis. For example, the overall brightness can be measured by the L ^* coordinate in the L ^* a ^* b ^* color space (International Commission on Illumination). Chromophore mapping, for example, melanin mapping, and melanin concentration can be used as indicators of overall skin tone. Average melanin can be calculated from chromophore map data. Furthermore, skin tone uniformity can also be determined by melanin uniformity, and melanin uniformity can also be calculated from chromophore map data. Suitable chromophore mapping techniques are discussed in the examples below.

  The term “facial skin surface” as used herein refers to one or more of the frontal, periorbital, cheek, perianal, chin and nasal skin surfaces.

  The term “conventional extract” as used herein refers to an extract produced by solvent extraction of a compound from plant material. The plant material may be dehydrated (ie, dry) plant material and / or non-dehydrated (eg, fresh or only partially dehydrated) plant material.

I. Compositions The present invention relates to various compositions, more specifically to compositions for application to the skin surface. The composition can take a wide variety of product forms including, but not limited to, solutions, suspensions, lotions, creams, gels, lotions, sticks, pencils, sprays, aerosols, ointments, cleansings. Liquid detergents and solid bars, shampoos and hair conditioners, pastes, foams, powders, mousses, shaving creams, wipes, strips, patches, electric patches, wound dressings and adhesive bandages, hydrogels, film-forming products, facial and skin Masks (which may or may not have an insoluble sheet), makeup, examples include foundations, eyeliners and eyeshadows. The form of the composition may follow the particular dermatologically acceptable carrier chosen if it is present in the composition.

  The compositions of the present invention are useful for reducing the hyperpigmented appearance of skin associated with melanin. As used herein, “reducing the visible appearance of hyperpigmentation of the skin” includes skin whitening. Skin lightening includes reducing, minimizing and / or eliminating (therapeutic) and / or melanin in the skin, including the hyperpigmented areas of the skin. Is involved in delaying, minimizing and / or preventing (preventive) the formation of. As used herein, “hyperpigmentation zone” means a localized zone of high melanin content, age-related spots, liver spots, spots full of spots, spot formation, melanosis, brown spots , Freckle, post-inflammatory pigmentation, or sun-induced pigmented spots.

A. Fig Serus Fraction Fig, or Fig genus, consists of many species and is found throughout the world. The fig genus should not be confused with the Ficus species, ie prickly pear cactus, Japanese name: Opuntia ficus-indica (L.) Mill, cactaceae (Barbera et al., Paste and Present Role of the Indian-fig Prickly-Pear (Opuntia ficus-indica (L.) Miller, Cactaceae) in the Agriculture of Sicily, Economic Botany 46 (1): 10-20, 1992). Notable species of the genus Fig include Ficus carica (general fig), Ficus religiosa (Indobodid, where Buddha was searching for the “truth”), Ficus elastica (Ficus elastica) ) Roxb. ExHorneum. (Indian rubber tree), Ficus behghalensis (Banyan tree), and Ficus racemosa (also known as glomerata; Fusanari fig).

  Figs are examples of multiple fruits that belong to the general category of fruits and have a unique “inside-out” structure with no inflorescences. These include aggregates of pebbles, which are fleshy hollow florets, each having a small opening at the apex, called a small hole. Small flowers gather on the inner wall and are not visible from the outside.

  As one of the oldest human foods known, fig species (ficus) have a long-standing safety profile. Fig fresh or dried fruit, tree bark, leaves, twigs, latex and young branches have been used for various purposes throughout history. Some of these historical uses include wounds, ulcers, cancerous growths, tumors, abscesses, gout, chronic cough, lung problems, chronic diarrhea, constipation, rheumatism, mania, hemorrhoids, diabetes, Treatment for vomiting, eczema, leprosy and warts.

  The fig serum fraction (hereinafter “FSF”) of the present invention is obtained from the cell juice of fresh fig leaves. Cell juice separated mechanically from fresh leaves is fractionated using pH adjustment, focused microwave radiation, centrifugation and sterile filtration to obtain a fig serum fraction free of pheophorbide and protein. The resulting fig serum fraction consists essentially of the cytoplasm of fig leaf cells. In certain embodiments, the fig cell juice may be F. benghalensis, F. carica, F. elastica, F. microcarpa, F. trigonata And from a Ficus species selected from the group consisting of combinations thereof.

  The composition of the present invention comprises a safe and effective amount of fig serum fraction. The composition contains FSF in an amount of 0.01% to 50%, in one embodiment, in an amount of 0.05% to 20%, in another embodiment, in another embodiment, relative to the weight of the composition. It can be contained in an amount of 0.2% to 10%. In yet another embodiment, the composition comprises 1% to 5% FSF, and in yet another embodiment, 1% to 3% FSF, relative to the total weight of the composition.

  The researchers found that when the fig serum fraction is applied topically to the skin, it has superior skin whitening effects, including whitening of hyperpigmented areas in mammalian skin compared to conventional fig extracts. It came to find out that it brings. The subject invention is not to be limited by theory, but is believed to operate by inhibition of processes involved in melanin production (melanogenesis), which can occur with, for example, UV or solar exposure It includes preventing stimulation of melanocytes by reactive oxygen / oxygen radicals (oxidative stress) that initiates the melanin production pathway in melanocytes.

Preparation of Fig Serous Fraction The method used to separate the compound from the plant, such as by extraction with a solvent, determines which compound is isolated. In accordance with the general principle of “similar ones dissolve well”, the choice of extraction solvent primarily determines the type and number of compounds resulting from any particular extraction technique. For example, polar compounds can be extracted by using a polar solvent, while nonpolar compounds can be extracted by using a nonpolar solvent. This results in the isolation of only a narrow range of compounds from the entire spectrum of compounds that may be present. Table 1x below summarizes the different types of compounds usually extracted using common solvents over a range of different polarities.

  The fig serum fraction of the present invention is not prepared by solvent extraction, but rather by separating the fresh cell juice found in plant leaves and the remaining plant bodies. This cell juice contains the complete spectrum of compounds found in fresh fig leaves since it has not been exposed to the extraction process. In contrast, the extract contains only a narrow range of compounds that can be separated using a particular solvent. Thus, the resulting fig serum fraction contains a much broader range of potentially active compounds than the extract contains.

  Furthermore, many extracts are not prepared from fresh leaves, but rather are prepared from dry plant material, which is subject to degradation due to dehydration. During dehydration, the cell wall is damaged and compound degradation occurs through mechanisms such as hydrolysis, oxidation, polymerization, Maillard reaction and isomerization. Thus, when extracting dry leaves, the resulting extract contains these degradation products that were not originally present in fresh plants. Furthermore, the extract contains only a range of compounds that can be isolated by a particular solvent. Therefore, the composition of the resulting dried leaf extract is very different from the composition of the fig serum fraction.

  The method for making the composition of the fig serum fraction comprises the steps of: (a) separating the fig cell juice from clean, fresh, unwilted fig leaves to obtain a fresh fig cell juice, comprising: A step in which no extraneous liquid is added before or during the separation, (b) filtering the fresh fig cell juice to obtain a cell juice containing no fibers, and (c) a cell juice containing no fibers To obtain a fig serum fraction. The fractionation step includes (1) removing chlorophyll from the cell juice containing no fiber to obtain supernatant I, and (2) removing pigment and protein from supernatant I to obtain a fig serum fraction. And (3) optionally adding a stabilizer to the fig serum fraction.

  In some embodiments, the stabilizer is selected from the group consisting of antioxidants, chelating agents, preservatives, and mixtures thereof. In certain embodiments, the stabilizer is selected from the group consisting of sodium metabisulfite, potassium sorbate, sodium benzoate, sodium methylparaben, pentylene glycol, and mixtures thereof.

  In certain embodiments, the step of removing chlorophyll from the cell juice without fibers comprises: (i) adjusting the pH of the cell juice without fibers to about 3 to adjust the pH of the cells without fibers. (Ii) heating the pH-adjusted cell-free cell juice to about 90 C over about 1 minute; (iii) adjusting the pH-adjusted cell juice without fiber Cooling to 30C, and (iv) separating the pH-adjusted cell-free cell juice into precipitate I and supernatant I.

  In certain embodiments, removal of pigment and protein from supernatant I comprises (i) adjusting the pH of supernatant I to 7.5 to form pH-adjusted supernatant I, (ii) separating pH-adjusted supernatant I into precipitate II and supernatant II, (iii) adjusting the pH of supernatant II to 3.6 to form pH-adjusted supernatant II, And (iv) separating the pH adjusted supernatant II into a precipitate III and a fig serum fraction.

  Prepare fig serum fraction using fresh fig leaves. The preparation methods herein maintain the integrity of the bioactive components that are essentially present in the fig leaf, resulting in a fig serum fraction that exhibits superior activity. During harvest and transport, care is taken to preserve leaf integrity so as to minimize environmental factors such as water loss and biological degradation. All steps are completed in the shortest possible time to minimize exposure of fresh leaves to sunlight, high temperatures, and other undesirable environmental factors.

  In certain embodiments, the fig leaf is a bengal bodyfish (F. benghalensis), a fig (F. carica), a ficus elastica, a banyan (F. microcarpa), a ficus trigonata, And a group of Ficus species consisting of combinations thereof.

  Harvesting, such as manual or mechanical cutting, is performed to avoid or minimize chopping, crushing, crushing, or other types of injury to the leaves. Harvesting and transport should be done to avoid wilting due to water loss. In one embodiment, fresh fig leaves are used to prepare a fig serum fraction containing at least 90% of their original moisture content, and in another embodiment, at least the original moisture content present at harvest. A fig serum fraction containing 95%, in another embodiment, containing at least 98% is prepared.

  Since up to 30% of the leaves of the fig plant can be harvested at once without adversely affecting the survival of the plant, ie subsequent leaf regrowth, harvesting from the same plant many times it can. Thus, fig plants grow throughout their full natural life, continue to be part of the surrounding ecosystem, and repeatedly provide leaf harvests to prepare cosmetic compositions that exhibit bioactivity. be able to. This harvesting method is preferred because it promotes the sustainability of natural resources.

  Less preferred, but less sustainable collection methods can also be used, such as using large-scale machine harvests that remove large amounts of plants and leave no viable parts for regrowth . However, even with this more invasive harvesting method, care is taken to minimize fig leaf injury. Fig leaf damage could result in leaf harvesting that intensifies microbial growth, water loss, oxidation, polymerization, isomerization and hydrolysis processes (ie, undesired catabolism processes). For example, in one embodiment of the invention, the fig plant is manually cut and collected as a whole plant. In another embodiment, plant leaves are cut using harvesting equipment.

  Minimize leaf delivery time to cut plant material processing facilities, as well as exposure to sunlight, high temperatures, and other undesired environmental factors to prevent the effects of the unwanted degradation processes described above Should. For example, in one embodiment of the present invention, the delivery time of the plant from the time of cutting until further processing does not exceed 30 minutes. In another embodiment, plants that have undergone long-distance transport are treated according to a post-cutting procedure, in which the fig leaf is immediately placed in a foamed polystyrene cooler containing a frozen gel pack bag to the processing facility. Including helping to maintain freshness and natural moisture content during overnight delivery. Similarly, other post-cutting procedures that achieve the results described above can be used.

The fig leaf is then gently washed before processing to remove soil particles and other debris. In one embodiment, washing is accomplished using a low pressure rinse for a short period of time under conditions that prevent cell juice from starting to release from the leaf, causing injury, or removing valuable components. . For example, in one embodiment of the present invention, fig leaf cleaning is achieved within 5 minutes using a water pressure of 1 kg / cm ² or less. The remaining wash water must not contain any green or yellow pigment. The absence of such a dye indicates that no secondary injury has occurred. Excess water is then removed from the washed leaves to keep the dry matter content as close as possible to the natural level.

  The washed fresh fig leaf is then mechanically separated to release the cell juice, which contains most of the intracellular material of the parenchymal cells, from the fiber-rich material mainly containing the cell walls. It is important not to add extraneous solvents (eg water, hexane, acetone, ethanol) during the separation process. As used herein, an “foreign solvent” is any solvent that is essentially absent from the plant material, but is contacted with the plant material for the purpose of separating (eg, extracting) the compound from the plant material. Means.

  Release of cell juice from the washed leaves involves crushing, maceration and pressing to obtain a liquid intracellular content (ie, cell juice) and separate the intracellular content from the fiber rich material. . In one embodiment, a hammer mill (Model VS35, Vincent Corporation, Tampa, FL) with a 5HP engine and a series of screens is used to grind the leaves so that the appropriate small sized leaf tissue particles are minimized. Within a period of time without significantly increasing the biomass temperature. In this embodiment, the hammer mill was set up to produce a maximum size macerated leaf particle of ≦ 2.0 centimeters during the treatment of ≦ 10 seconds, in this case the macerated fresh leaf The temperature increases only by ≦ 2 ° C. or less.

As described above, exposure of ground and macerated fig leaves is minimized to prevent the effects of unwanted catabolic processes. Fig leaves are processed in a short time without significantly increasing the temperature. Immediately after grinding and maceration, the fig leaf is pressed to obtain cell juice from the fresh macerated leaf. In one embodiment, this is accomplished using a horizontal continuous screw press (Compact Press “CP-6”, Vincent Corporation, Tampa, FL) equipped with a cone supported by compressed air. In this embodiment, the pressure on the cone is maintained at a level of 15 kg / cm ² or higher, the screw speed is 12 rpm, and the increase in cell juice temperature is 5 ° C. or less.

  This treatment yields a fiber-rich material and cell juice. The remaining small fiber particles are then removed from the cell juice. This is because these fiber particles can absorb valuable cell juice components and can also block the equipment hoses and pumps. For example, these particles can be removed by filtration or low speed centrifugation. In one embodiment, these particles are removed by purification using a continuous flow centrifuge (Model 12-413V, AML Industries, Inc., Hatboro, Pa.) With a fully automatic discharge unit. At a flow rate of 2 liters / minute, the retention time for cell juice purification at ≦ 2,250 g is ≧ 100 seconds. This regimen produces a cell juice that does not contain fibers. The precipitate containing the small fiber particles is collected and combined with the remaining, fiber rich material produced after fresh leaf pressing.

  If desired, at this point the cell juice can be frozen and stored in an airtight, non-reactive container for later processing. In one embodiment, the cell juice is placed without delay in a tightly closed 15 liter rectangular HDPE container and frozen at −30 ° C. The solid frozen cell juice is kept at this low temperature for further use.

  The frozen cell juice can then preferably be converted back to a liquid state by fluidization over a short period of time (eg, 2 minutes or less) while minimizing the increase in cell juice temperature during fluidization. Fasten (for example, 20C or less). A short fluidization period and low temperature rise minimize both degeneration and oxidative damage to the cell juice. This results in a cell juice having physiochemical and biochemical properties that are essentially the same as those measured before freezing.

  Cell juice is composed of three major types of components: (i) membrane-bound chloroplast, mitochondria, endoplasmic reticulum, nucleus, lysosome, peroxisome, vacuole, Golgi; and (ii) non-membrane-bound Ribosomes, microtubules; and (iii) components not related to the above groups, such as the cytoplasm. Due to the presence of organelles and their fragments, and unwanted dyes and proteins in the cell juice, including but not limited to color, solubility, transparency, stability and in vitro activity, Fractionation is necessary to produce personal care ingredients with the desired combination of functional properties.

  Various treatments are used to fractionate cell juice, including pH adjustment, focused microwave radiation, centrifugation and vacuum filtration. The resulting isolated cell juice serum fraction is then stabilized with preservatives and antioxidants to produce the final fig serum fraction.

  The pH of the cell juice is adjusted to ≧ 3.0 (pH adjustment 1). In one embodiment, the pH of the cell juice close to neutral (7.0) is adjusted using a titration method utilizing 5.0 N hydrochloric acid (HCl) to adjust the pH of the cell juice to ≧ 3.0. (PH adjustment 1).

  Chlorophyll is then removed from the conditioned cell juice. In addition to the desirable absence of this pigment in cosmetic ingredients, chlorophyll may be converted to pheophorbide, which is a toxic compound (Bergstrom, LC, Vucenik, I., Hagen , IK, Chernomorsky SA, Poretz RD In-vitro photocytotoxicity of lysosomotropic immunoliposomes containing pheophorbide a with human bladder carcinoma cells, J. Photochem. Photobiol., 24, 1, 17-23, 1994), considered to be involved in skin irritation (Kato T., Yamada K., Relationship between appearance of photosensitization and total pheophorbide level in spirulina powder, J. Food Hyg. Soc. Japan, 36, 632-634, 1995).

  In one embodiment, removal of chlorophyll is achieved by heating the conditioned cell juice, followed by cooling and subsequent separation of the precipitate from the supernatant. In certain embodiments, the conditioned cell juice is processed without delay by focused microwave radiation using a frequency of 2,450 MHz. During this focused microwave treatment (FMP), the temperature of the cell juice is temporarily increased to 90 ° C. and kept at this temperature for 1 minute, and then the temperature of the cell juice is immediately decreased to ≦ 30 ° C. The treated cell juice is then rapidly separated using a continuous flow centrifuge CEPA LE (Carl Padberg Zentrifugenbau GmbH, Germany) with a retention time of 15,000 rpm and ≧ 30 seconds. Separation of the treated cell juice yields a green colored paste-like precipitate ("Precipitate I") and a light brown colored liquid supernatant ("Supernatant I") that emits a slight opalescence. . This supernatant I is used for further fractionation.

  Supernatant I is further processed to greatly remove other undesired compounds, including brown pigment and residual protein. This treatment includes pH adjustment and separation. Adjust the pH of the supernatant I to increase the pH to about 7.5 (pH adjustment 2). In one embodiment, pH adjustment 2 is accomplished by a titration method utilizing 50% sodium hydroxide (NaOH) to increase the pH of cell juice supernatant I from about 3.0 to about 7.5 ( pH adjustment 2). As a result of pH adjustment 2, a darker material is produced and the opalescence is enhanced, which is then purified by separation. In one embodiment, purification is achieved by using a continuous flow centrifuge CEPA LE (Carl Padberg Zentrifugenbau GmbH, Germany) with a retention time of 15,000 rpm and ≧ 30 seconds. As a result of this separation, a brownish colored paste (precipitate II) and a brown colored supernatant (supernatant II) that slightly emits milky white light are obtained.

  Next, the pH of the supernatant II is adjusted (pH adjustment 3) and filtered. Adjust the pH of supernatant II to reduce the pH to about 3.6 (pH adjustment 3). In one embodiment, Supernatant II is subjected to titration utilizing 5.0 N hydrochloric acid (HCl) to reduce the pH value to about pH 3.6 (pH adjustment 3). Such a treatment results in a slight increase in opalescence but a supernatant II that is titrated to a lighter pH. The pH adjusted supernatant II is processed using sterile filtration through a membrane with a pore size of 0.2 micrometers. The resulting filtrate is a light colored clear fig serum fraction (FSF). FSF consists essentially of the cytoplasm of fig leaf cells.

  Further stabilization of the serum fraction can be achieved by adding antioxidants, stabilizers, chelating agents and preservatives. In one embodiment, the following additives are added to the fig serum fraction: 0.2% sodium metabisulfite, 0.1% potassium sorbate, 0.1% sodium benzoate, and 0.1% methylparaben sodium. Add to. In this embodiment, the mixture is incubated (≧ 30 minutes) until complete solubilization of the additive is achieved. 1.9% pentylene glycol was then added to the mixture.

FSF Properties The resulting FSF demonstrates the properties that make it desirable for use as a cosmetic ingredient. These properties are stable, water soluble, undesired materials such as the absence of pheophorbide and protein, lighter colors, higher solids content, and higher levels of desirable compounds such as phenylalanine Including doing.

  Stability studies indicate that the cosmetic ingredients produced by these methods from FSF are stable for at least 6 months at room temperature. As used herein, “stable” refers to the physical properties of the composition over a specified period of time when stored at STP (standard temperature and pressure: 25 ° C., 1 atm) in dark, dry areas. Means that there is no significant change in the mechanical or chemical properties. These properties include color and chemical composition. The FSF and compositions comprising it are stable in some embodiments for at least 6 months, in other embodiments for at least 12 months, and in still other embodiments for at least 24 months. In certain embodiments, the composition is stable for 6-24 months, 12-24 months, or 6-12 months.

Water solubility The FSF of the present invention is also water soluble. As used herein, “water soluble” means that FSF is miscible with water (in STP) in any ratio. Because FSF is water soluble, FSF provides greater flexibility when formulating a composition comprising figs. For example, for water-based formulations, a non-sticky feel, a desirable coatability, a light skin feel, and ease of removal from the skin surface (eg, rinsing) are often desired by consumers.

  However, conventional fig extracts that do not dissolve in water are extracted using solvents, resulting in non-uniform active substance concentrations throughout the phase separation, sedimentation, crystallization, and water-based compositions. May cause difficulties in the formulation. In order to overcome the formulation problems associated with active substances that do not dissolve in water, more complex formulations, for example emulsions (in this case, oily and / or oily materials are usually introduced into the composition) ) Is usually used. This results in sticky, heavy and / or viscous skin feel compositions, compositions that are not easily removed from the skin surface, and more expensive and / or complex manufacturing processes. In many cases, these formulations may also prevent delivery of the active substance to the skin.

  However, because FSF is sufficiently water-soluble, FSF can be incorporated into water-based formulations without causing the problems mentioned above with conventional fig extracts that do not dissolve in water. . This results in greater pharmaceutical flexibility and thus can result in fig compositions having superior consumer-desired attributes.

  Furthermore, because FSF is sufficiently water soluble, FSF is more biologically available than solvent extracts that are not sufficiently water soluble. This makes the delivery of active components derived from FSF to the skin more effective.

Furthermore, many measurements of the potential biological activity of conventional solvent extracts are not feasible due to their insolubility in water. For example, in this study, IC ₅₀ values of solvent extracts could not be measured because they are not water soluble.

Safety / Allergenicity FSF is also substantially free of pheophorbide and proteins, which are materials commonly found in plants, including figs. These materials are known to produce safety concerns, such as toxicity and / or allergic reactions, in susceptible individuals. At levels normally found in plants, these materials are usually not a concern. However, when plant material is concentrated, such as by processing, the relative concentration can show a dramatic increase, creating safety concerns. Therefore, compositions that do not contain these materials are highly preferred.

  Pheophorbide is a pigment compound that is a degradation product of chlorophyll. These pigments are also known to be biological toxins and skin photosensitizers in addition to discoloring the product. (Bergstrom, LC, Vucenik, I., Hagen, IK, Chernomorsky SA, Poretz RD In-vitro photocytotoxicity of lysosomotropic immunoliposomes containing pheophorbide a with human bladder carcinoma cells, J. Photochem. Photobiol., 24, 1, 17-23, 1994); (Kato T., Yamada K., Relationship between appearance of photosensitization and total pheophorbide level in spirulina powder, J. Food Hyg. Soc. Japan, 36, 632-634, 1995).

  As shown in FIG. 2, the conventional fig extract (of Example 3) contains a slow eluting (ie, more hydrophobic) compound that is not detected in the FSF (Example 1). As shown in FIG. 3, the LC / UV chromatograms of the slow-elution compounds of the conventional fig extract and their corresponding extracted ion chromatograms identified these compounds as pheophorbide. (See Example 5)

  Proteins can cause protein contact dermatitis in susceptible individuals, including proteins in plants such as figs. Symptoms such as acute urticaria-like or blistering rash on the skin, often accompanied by pruritus, burning and / or tingling, immediately following contact with the causative proteinaceous material May experience. (V. Janssens et al., “Protein contact dermatitis: myth or reality?”, British Journal of Dermatology, 1995; 132: 1-6). Therefore, it is highly desirable that skin care materials contain as little protein as possible.

  FSF was tested for total protein content using the Kjeldahl method (Example 1, Table 3). No protein was detected in the FSF. As used herein, “substantially free of protein” means a total protein content of less than 1% (0% to 1%) when using the Kjeldahl method. The protein content is 0% to 1% of FSF in some embodiments, 0% to 0.5% in other embodiments, and 0% to 0.25 in other embodiments. %.

Color / Color Stability FSF has a lighter color than conventional fig extract. The FSF has a Gardner chromaticity value of less than 8, and in some embodiments, a Gardner chromaticity value of less than 7.5. Certain embodiments have a Gardner chromaticity value of 5-8, other embodiments have a Gardner chromaticity value of 6-8, and other embodiments have a Gardner color of 6.5-8. Has a degree value. FIG. 8 shows the difference between the color of the FSF and the color of the dried leaf fig extract after a 14-day accelerated degradation study. FIG. 10 shows the color difference of 0.55% FSF in a vehicle with varying levels of stabilizer / preservative. FIG. 9 is a calibration chart constructed for analysis of the accelerated degradation study of FIG.

  The researchers have identified several interesting constituents in conventional extracts. These components are not present in the FSF and / or are present at a much lower level and could cause differences in color and color stability between the two. For example, FIG. 4 shows that conventional extracts contain higher amounts of components that appear to be flavonol glycosides. Flavonol glycosides can be combined with tannins (tannins are found in both FSF and conventional extracts) to form polymeric dyes. Thus, higher levels of flavonol glycosides could result in the formation of high levels of pigment compounds in conventional extracts. In addition, the polyphenolic structure of flavonoids and tannins is then highly sensitive to factors such as oxidation, heat and light, which is a potential cause of the increase in the presence of pigments in conventional extracts over time. Can be.

Solid content The solid contains the biologically active portion of the FSF or extract. Therefore, the higher the solid content, the higher the botanical activity. FSF has a higher solids content compared to conventional water soluble fig extracts. The FSF has a solid (dry matter) content of greater than 5% with respect to the weight of the FSF, and in certain embodiments, a solid (dry matter) content of 5% to 20%, or 5% to 10%. Have.

Bioactivity of FSF FSF exhibits at least four different mechanisms of action that are recognized to control the production of pigmentation in the skin. These mechanisms are tyrosinase inhibition, trypsin inhibition, COX-2 inhibition and antioxidant activity. The FSF, in one embodiment, exhibits at least one of the following pigmentation reducing activities, and in other embodiments, exhibits at least two of the following pigmentation reducing activities, and alternatively, at least 3 Shows: Tyrosinase inhibition IC50 (% DM), 0.003-0.06; Trypsin inhibition IC50 (% DM), 0.02-0.5; COX-2 inhibition IC50 (% DM), 0.02-1 And antioxidant superoxide capture capacity, as measured by DPPH assay (1 / x DM), 1-15, and / or as measured by ORAC assay (1 / x DM), 0.2-5. As used herein, “DM” is a dry substance (“solid”), “ORAC” is the ability to absorb oxygen radicals, and “DPPH” is a measure of the ability to scavenge free radicals.

  Furthermore, as demonstrated by the B16 melanin inhibition assay of Example 6, FSF is more effective in inhibiting melanin production than conventional solvent extract of fig dried leaves. For example, FSF produced a degree of inhibition of melanin synthesis (48.1% inhibition of melanin) at a concentration of 0.01 more than twice as much as conventional extracts (23% inhibition of melanin). .

  Accordingly, the present invention provides a method for controlling melanogenesis (ie, melanin suppression) in the skin. Various forms of hyperpigmentation (for example, freckles, age-related spots, liver spots, spots full of spots, mottled pigmentation, etc.) involving the concentration of melanin in the skin are present in melanocytes in the epidermis And is thought to result from changes in keratinocytes. Melanocytes are located at the base of the epidermis and, as they age, lose their normal control processes and produce excess pigment. This excessive production results in the formation of dense perinuclear aggregates of melanin in keratinocytes within the epidermis, resulting in hyperpigmented areas.

  Conventional therapies for hyperpigmented skin involve the application of specific agents that whiten the skin that inhibit melanogenesis. The mechanism of action for these materials proposed in the art is inhibition of tyrosinase and / or inhibition of other steps in melanin synthesis. Tyrosinase is present in melanosomes in epithelial melanocytes and catalyzes a directed step in the formation of melanin from tyrosine. (See Goldsmith, LA, PHYSIOLOGY, BIOCHEMISTRV, AND MOLECULAR BIOLOGY OF THE SKIN, Oxford University Press, pages 873-903, N.Y., 1991). Tyrosinase catalyzes the hydroxylation of tyrosine and the oxidation of DOPA to DOPA quinine. Thus, when the inhibitor binds to the active site of tyrosinase, a decrease in melanogenesis occurs. See generally Prota, G., Melanins and Melanogenesis, Academic Press, Inc. (San Diego, 1992).

  Oxidative processes are involved in non-enzymatic steps of melanin production. Conversion of DOPAquinone to melanin occurs via non-enzymatic or spontaneous chemical reactions, some of which involve reactive oxygen species (ROS) or oxygen radicals. Oxidative stress on melanocytes, such as by stimulation of reactive oxygen / oxygen radical species (which can occur with exposure to UV or sunlight, for example) initiates a melanin production pathway within melanocytes. Various antioxidants / radical scavengers have been used to help interfere with these processes and thus achieve the benefits of whitening the skin.

  It is known that metabolites derived from arachidonic acid act as potent inflammatory mediators in the skin, especially in response to environmental insults such as UV, smog and other such stimulants. It has been. In this pathway, membrane phospholipids are converted to arachidonic acid (AA) by phospholipase A2. Once formed, AA is exploited by one of two competing biological pathways, either the cyclooxygenase (COX) pathway or the 5-lipoxygenase pathway. The most relevant enzyme in the COX inflammatory pathway is COX-2, which catalyzes the conversion of arachidonic acid to PGH2, which is rapidly converted to prostaglandins such as PGE2. Sex molecule. Prostaglandins are autocrine or paracrine that act as local messengers involved in eliciting an inflammatory response at the site of stimulation.

  The absorption of the fig serum fraction into the skin inhibits COX-2 and prevents the metabolites derived from arachidonic acid from being converted to prostaglandins. The net effect is a reduction in both basal and induced prostaglandin pools. Reduction of prostaglandin levels results in both a direct reduction of the inflammatory response and a reduction in all downstream messenger activity that results. Two of these messenger activities include activation of melanin synthesis in melanocytes and inhibition of collagen production in fibroblasts.

  Prostaglandins are known to stimulate melanocytes by increasing the amount of tyrosinase, an enzyme involved in melanin production. Stimulation of melanocytes and overproduction of melanin results in hyperpigmentation, which is observed as darkening of the skin area. The discoloration caused by inflammation and thus as a result of direct stimulation of prostaglandins to melanocytes is called hyperpigmentation after inflammation. The reduction in prostaglandin production caused by inhibition of COX2 by the fig serum fraction will result in reduced melanin production and a more evenly colored skin.

Prostaglandin PGE ₂ is prominent in reducing type I and / or type III collagen synthesis in a variety of cells, including human skin fibroblasts, rat glomerular stromal cells and liver astrocytes It has been shown to have an effect [ref]. This supports that an increase in PGE ₂ levels in response to inflammation results in inhibition of collagen synthesis, as type I and type III are the prominent forms of collagen that make up the dermal dermis. AA and PGE ₂ have been shown to have an inhibitory effect on collagen synthesis. Adding natural COX-2 inhibitors, including the omega-3 fatty acids EPA and DHA, counteracts the inhibition of collagen synthesis caused by PGE ₂ , resulting in a net increase in collagen synthesis Was possible. Thus, the fig serum fraction appears to improve skin texture by inhibiting COX2, which inhibits the formation of prostaglandins that cause collagen inhibition.

  When the fig serum fraction is applied topically to the skin, it achieves superior skin lightening effects, including whitening of hyperpigmented areas in mammalian skin, compared to conventional fig extracts. I came to find it unexpectedly. Furthermore, analytical studies have also shown that the fig serum fraction of the present invention interferes with melanin synthesis through multiple mechanisms of action, affecting both enzymatic and non-enzymatic pathways. FSF led to enhanced bioactivity compared to conventional fig extract. The physiological activity includes one or a combination of enzyme inhibitory activity, free radical scavenging activity, antioxidant activity, and melanin synthesis inhibitory activity. Enzyme inhibitory activity includes, but is not limited to, one or a combination of the inhibitory activities of tyrosinase, elastase, trypsin, and cyclooxygenase-2 (“COX-2”). Antioxidant activity includes, but is not limited to, oxygen radical absorption ability.

  The investigators have also shown that traditional fig extracts can, for example, provide benefits for hyperpigmentation, but these conventional extracts are not as effective as these, The properties shown by are less suitable for use in cosmetic compositions and are therefore less desirable.

  As shown in FIG. 5, FSF has higher levels (about 10 ×) of catechins and associated condensed tannins. Condensed tannins such as catechin (proanthocyanidins) are a class of flavanols. Proanthocyanidins are essentially polymer chains of flavonoids such as catechins. Tannins are thought to function as biological antioxidants (free radical scavengers) in the human body and are widely used to combat oxidative damage to the skin, for example, damage caused by aging. It is considered. Furthermore, antioxidants can help protect against the effects of internal and environmental stresses, such as tobacco smoking and contamination, and can also support normal body metabolic processes. (Kehrer, J.P., Crit. Rev. Toxicol, 1993, 23, 21). FIG. 7 also shows that FSF contained higher levels of free tyrosine, phenylalanine and tryptophan, which are essential amino acids. However, as shown in FIG. 6, the three caffeoylquinic acid isomer levels detected did not appear to differ substantially between the two samples.

B. Skin Tone Agent In some embodiments, it may be desirable to include a skin tone agent in combination with FSF in the composition. Skin tone agents can be included to further improve the overall skin tone. The compositions of the present invention contain up to about 50%, 40%, 30%, 20%, 10%, 5% or 3% skin tone agent, if present, relative to the weight of the composition. The composition of the present invention contains at least about 0.001%, 0.01%, 0.1%, 0.2%, 0.5% or 1% skin tone agent, relative to the weight of the composition. If you want to contain. Suitable skin tone agents, including suitable ranges of from about 0.1% to about 50%, from about 0.2% to about 20%, or from about 1% to about 10%, as appropriate ranges, by weight of the composition Arbitrary combinations of the lower limit and the upper limit of are mentioned. The amounts listed herein should only be used as a guide. This is because the efficacy of skin tone agents varies considerably and their optimal amount depends on the particular active substance chosen.

  Suitable skin tone agents include, but are not limited to, sugar amines, vitamin B3 compounds, arbutin, deoxyarbutin, 1,3-dihydroxy-4-alkylbenzenes such as hexyl resorcinol, sucrose dilaurante, Bacthiol (4-[(1E, 3S) -3-ethenyl-3,7-dimethyl-1,6octadienyl] phenol or monterpene phenol), pyrenoine (available from Biotech Marine, France), millet ( panicum miliaceum) seed extract, arlatone diacid, cinnamic acid, ferulic acid, achromaxyl, methyl nicotinamide, oil-soluble licorice extract, folic acid, undecylenic acid (ie undecenoic acid), zinc undecylenate, thiamine (vitamin) B1) and its hydrochloride salt, L-tryptophan, extract of leaves of sunflower and vitis vinifera (carp), carnosine (i.e. dragosine), methyl gentisate, 1 , 2-hexanediol and 1,2-octanediol (ie, combination sold by Symrise AG, Germany as Symdiol 68), inositol, decylenoylphenylalanine (eg, under the trade name Sepiwhite, Seppic, France) And retinoids, including koijic acid, hexamidine compounds, salicylic acid, and retinyl and retinyl propionate.

  In certain embodiments, the additional skin tone agent is selected from vitamin B3 compounds, sugar amines, hexamidine compounds, salicylic acid, 1,3-dihydroxy-4-alkylbenzenes, such as hexyl resorcinol, and retinoids. As used herein, “vitamin B3 compound” has the formula:

[Wherein R is —CONH ₂ (ie, niacinamide), —COOH (ie, nicotinic acid), or —CH ₂ OH (ie, nicotinyl alcohol); derivatives thereof; and any of the foregoing Is a salt of]. As used herein, “sugar amine” includes isomers and the tautomers thereof, as well as salts (eg, HCl salts) and derivatives thereof. Examples of sugar amines include glucosamine, N-acetylglucosamine, mannosamine, N-acetylmannosamine, galactosamine, N-acetylgalactosamine, isomers thereof (eg, stereoisomers), and salts thereof (eg, HCl salts) ). As used herein, a “hexaminide compound” has the formula:

Where R ¹ and R ² are optional or are organic acids (eg, sulfonic acids, etc.). In one embodiment, the hexamidine compound is hexamidine diisethionate.

C. Anti-inflammatory agents Hyperpigmentation may result from skin irritation. Hyperpigmentation, and more specifically, transient inflammatory events that cause hyperpigmentation after inflammation include, but are not limited to, pressure ulcer lesions, hair grown inside, scratches, insect bites, Surfactant damage, allergens, and short-term UV exposure. Pro-inflammatory hyperpigmentation, including post-inflammation hyperpigmentation, can be managed by incorporating anti-inflammatory agents in the compositions of the invention. The compositions of the present invention contain up to about 20%, 10%, 5%, 3% or 1% anti-inflammatory agent, if present, relative to the weight of the composition. The composition of the present invention has at least about 0.001%, 0.01%, 0.1%, 0.2%, 0.3%, 0.5% or 1% anti-inflammatory with respect to the weight of the composition The agent is included if present. A suitable range includes any combination of lower and upper limits. Suitable anti-inflammatory agents include, but are not limited to, nonsteroidal anti-inflammatory agents (including but not limited to ibuprofen, naproxen, flufenamic acid, etofenamate, aspirin, mefenamic acid, meclofenamic acid, piroxicam and felbinac NSAIDs), glycyrrhitenic acid (also known as glycyrrhizinic acid, glycyrrhetinic acid, and glycyrrhetinic acid glycosides) and dipotassium glycyrrhetinic acid, glycyrrhetenic acid Licorice extract, bisabolol (eg, alpha bisabolol), manjistha (extracted from Rubia cordifolia, in particular, Rubia cordifolia) and guggal (into Mirran, in particular) , Gugu (Extracted from Commiphora mukul), cola extract, chamomile, red clover extract, as well as mutual coral extract (extract from goats), any of the above derivatives, and mixtures thereof Is mentioned.

D. Sunscreen Active Substances The compositions of the subject invention can include one or more sunscreen active substances (or sunscreen agents) and / or ultraviolet light absorbers. As used herein, “sunscreen active” collectively includes sunscreen actives, sunscreens and / or ultraviolet light absorbers. Sunscreen actives include both sunscreen agents and physical sunblocks. Sunscreen actives may be organic or inorganic. Examples of suitable sunscreen actives are disclosed as “sunscreens” in the Personal Care Product Council's International Cosmetic Ingredient Dictionary and Handbook, Thirteenth Edition. Particularly suitable sunscreen actives are 2-ethylhexyl p-methoxycinnamate (commercially available as PARSOL ™ MCX), 4,4′-t-butylmethoxydibenzoyl-methane (PARSOL ™ 1789) Commercially available), 2-hydroxy-4-methoxybenzophenone, octyldimethyl-p-aminobenzoic acid, digalloyl trioleate, 2,2-dihydroxy-4-methoxybenzophenone, ethyl-4-benzoate aminobenzoate (bis ( Hydroxypropyl)), 2-ethylhexyl 2-cyano-3,3-diphenylacrylate, 2-ethylhexyl salicylate, glyceryl p-aminobenzoate, 3,3,5-tri-methylcyclohexyl salicylate, menthyl anthranilate, p- Dimethylaminobenzoic acid or amino Benzoic acid ester, p-dimethyl-amino-benzoic acid 2-ethylhexyl, 2-phenylbenzimidazole-5-sulfonic acid, 2- (p-dimethylaminophenyl) -5-sulfonebenzooxazonic acid, octocrylene, zinc oxide Benzylidene camphor and derivatives thereof, titanium dioxide, and mixtures thereof.

  In one embodiment, the composition may comprise about 1% to about 20%, alternatively about 2% to about 10% sunscreen active, based on the weight of the composition. The exact amount will vary depending on the sunscreen active chosen and the desired UV protection factor (SPF), which is within the knowledge of those skilled in the art.

E. Optional Components The compositions of the present invention can contain them, provided that variations in the variety of other ingredients to the benefit of the present invention are acceptable. The compositions of the present invention are optionally from about 0.0001% to about 50%, from about 0.001% to about 20%, or alternatively from about 0.01% to about 10%, by weight of the composition These components, if present, can be included. The amounts listed herein should only be used as a guide. This is because the potency of optional components used in the composition varies considerably, so their optimal amount will depend on the particular active substance chosen. Accordingly, the amount of some optional components useful in the present invention may be outside the ranges listed herein.

  The optional components should be suitable for use in contact with human skin tissue without excessive toxicity, incompatibility, instability, allergic response, etc. when incorporated into the composition. The composition of the present invention comprises optional components, such as anti-decubitus active substances, exfoliation active substances, anti-cellulite agents, chelating agents, flavonoids, sun tanning active substances, non-vitamin antioxidants and radical scavengers Hair growth regulators, anti-wrinkle actives, anti-atrophy actives, minerals, plant sterols and / or plant hormones, N-acyl amino acid compounds, antibacterial or antifungal actives, and other useful skin care actives Which are described in more detail in US Application Publication Nos. US2006 / 0275237A1 and US2004 / 0175347A1.

  The Personal Care Product Council's International Cosmetic Ingredient Dictionary and Handbook, Thirteenth Edition, lists a wide variety of non-limiting cosmetic and pharmaceutical ingredients commonly used in the skin care industry, Optional component suitable for use in the compositions of the present invention. Examples of these component classes include abrasives, absorbents, aesthetic components, ie fragrances, pigments, colorants / colorants, essential oils, anti-hardening agents, anti-foaming agents, antibacterial agents, binders , Biological additives, buffering agents, bulking agents, chelating agents, chemical additives, colorants, cosmetic astringents, cosmetic biocides, denaturants, astringents as drugs, emollients, pharmacy Necessary analgesics, film formers or film materials, opacifiers, pH adjusters, preservatives, sprays, reducing agents, scavengers, skin cooling agents, skin protectants, thickener viscosity modifiers, vitamins, and their The combination of these is mentioned.

F. Dermatologically Acceptable Carrier The composition of the present invention may also include a dermatologically acceptable carrier for the composition, which may be referred to as a “carrier”. The phrase “dermatologically acceptable carrier” as used herein means that the carrier is suitable for topical application to keratinous tissue, has good aesthetic properties, and is active in the composition. Means that it does not cause any unreasonable safety or toxicity concerns. In one embodiment, the carrier is about 50% to about 99%, about 60% to about 98%, about 70% to about 98%, or alternatively about 80% to about 95% by weight of the composition. Exists at level.

  The carrier can take a wide variety of forms. Non-limiting examples include simple solutions (eg, solutions based on aqueous solvents, organic solvents or oils), emulsions, and solid forms (eg, gels, sticks, flowable solids or amorphous materials). It is done. In certain embodiments, the dermatologically acceptable carrier takes the form of an emulsion. Emulsions can generally be classified as having a continuous water phase (eg, oil-in-water and water-in-oil-in-water) or a continuous oil phase (eg, water-in-oil and oil-in-water-in-oil). . The oil phase of the present invention can include silicone oils; hydrocarbon oils, non-silicone oils such as esters, ethers, and the like, and mixtures thereof.

  The aqueous phase typically contains water. However, in other embodiments, the aqueous phase includes, but is not limited to, water soluble humectants, conditioning agents, antibacterial agents, water retention agents, and / or other water soluble skin care actives, Components other than water can be included. In one embodiment, the non-aqueous component of the composition includes a water retention agent such as glycerin and / or other polyols. However, it should be appreciated that the composition may be substantially anhydrous (ie, less than 1% water) or completely anhydrous.

  Appropriate carriers are selected to obtain the desired product form. Further, the solubility and dispersibility of the components (eg, FSF, sunscreen active, additional components) may determine the form and characteristics of the carrier. In one embodiment, an oil-in-water or water-in-oil emulsion is preferred.

  The emulsion can further comprise an emulsifier. The composition can include any percentage of an emulsifier suitable to fully emulsify the carrier. Suitable weight ranges include from about 0.1% to about 10% or from about 0.2% to about 5% emulsifier of the composition. The emulsifier can be nonionic, anionic or cationic. Suitable emulsifiers are disclosed, for example, in US Pat. No. 3,755,560, US Pat. No. 4,421,769, and McCutcheon's Detergents and Emulsifiers, North American Edition, pages 317-324 (1986). Suitable emulsions can have a wide range of viscosities, depending on the desired product form.

  The carrier may further comprise a thickening agent, as is well known in the art to provide a composition having suitable viscosity and rheological characteristics.

G. Exemplary Compositions Non-limiting examples of compositions of the present invention are shown below. These examples are given for purposes of illustration only and those skilled in the art will recognize that many variations thereof are possible without departing from the spirit and scope of the invention. The examples should not be construed as limiting the invention. In the examples, all concentrations are stated as weight percent unless otherwise stated, and trace amounts of materials such as diluents, fillers, etc. can be excluded. Accordingly, the described formulations comprise the described components and any minor amounts of materials associated with such components. As will be apparent to those skilled in the art, the choice of these trace amounts of material will vary depending on the physical and chemical characteristics of the particular components selected to make the invention described herein.

  All examples can be used to treat or improve the appearance of one or more hyperpigmented stains. In addition, the present invention provides for local treatment of one or more hyperpigmented spots with a first composition (eg, Example A or B), and a second composition (eg, Example C, D and E) may also relate to regimens involving wider or general facial skin treatment, which can be applied before or after local treatment to improve skin tone across the face .

  The compositions of the present invention are generally prepared by conventional methods such as methods known in the art of making topical compositions. Such methods typically involve mixing the components in a relatively uniform state in one or more steps, which may or may not involve heating, cooling, applying a vacuum, etc. There may not be. Typically, the emulsion is prepared by first mixing the aqueous phase material separately from the fatty phase material and then combining the two phases as necessary to obtain the desired continuous phase. . The composition is preferably prepared to optimize the stability (physical stability, chemical stability, light stability) and / or delivery of the active material. This optimization is appropriate for pH (eg, less than 7), complexed with active agents, and thus the elimination of materials that may have undesirable effects on stability or delivery (eg, The elimination of contaminating iron), the use of approaches to prevent complex formation (eg packaging of suitable dispersing agents or double compartments), the use of suitable light stability approaches (eg tanning) Stop / sunblock integration, use of opaque packaging) and the like.

  The compositions of the present invention preferably contain 0.01% or about 0.01% to 10% or about 10% fig serum fraction, more preferably 0.05% or about 0.05. % To 5% or about 5%, most preferably 0.1% or about 0.1% to 5% or about 5%, eg 2% fig serum fraction.

H. Method for Whitening Skin The composition of the present invention is useful for whitening mammalian skin (especially human skin, more particularly facial and hand skin). The composition is particularly useful for whitening the hyperpigmented areas of the skin.

  A method of whitening the skin (including hyperpigmented areas) involves topically applying to the skin a safe and effective amount of the composition of the present invention. In one embodiment, the cosmetic composition of the present invention may comprise 0.01% to 10% fig serum fraction. The amount of composition to be applied, the frequency of application and the duration of use will depend on the level of the fig serum fraction and / or other components of a given composition and, for example, the pigmentation of the skin present in the subject. It varies greatly depending on the level and whitening level desired in light of the rate of further pigmentation of the skin.

  In a preferred embodiment, the composition is applied to the skin continuously over time. “Long-term continuous topical application” refers to a long term, preferably at least about 1 week, more preferably at least about 1 month, even more preferably at least about 3 months, during the subject's lifetime. Even more preferably, it means that the composition is topically applied substantially continuously for at least about 6 months, even more preferably for at least about 1 year. Although benefits may be obtained after use of various longest periods (eg, 2, 5, 10 or 20 years), it is preferable to continue to apply continuously over the life of the subject. Typically, application will occur approximately 1 or 2 times per day over such long periods, although the rate of application may vary, for example, from about once a week to up to about 3 or more times per day Can do.

  A wide range of amounts of the composition of the present invention can be utilized to provide the benefit of whitening the skin. The amount of composition presented, usually applied per application, is about 0.1 mg / cm 2 skin to about 10 mg / cm 2 skin. A particularly useful application amount is about 2 mg per cm 2 of skin.

  The term “topical application” as used herein means to apply or apply the composition of the present invention onto the skin surface. Preferred compositions of the present invention take the form intended to remain over a long period of time (eg, several hours) in contact with the skin after topical application, eg, typically a cream, Use lotion or moisturizing liquid.

  The method of whitening the skin preferably takes the form of a skin lotion, cream, cosmetic, etc. intended to remain on the skin and provide some aesthetic, prophylactic, therapeutic or other benefit. By applying the composition to be applied topically. After application of the composition to the skin, the composition is preferably at least about 15 minutes, more preferably at least about 30 minutes, even more preferably at least about I hours, most preferably at least several hours, such as Remain on the skin for up to about 12 hours.

  The compositions of the present invention also include mammalian skin (especially human skin), including signs of skin aging, and visible and / or tactile skin discontinuities that accompany skin aging. It is also useful for more general control of the condition of the face and / or the skin of the hands, more particularly. Such control includes prophylactic and / or therapeutic control. Control of the skin condition involves topically applying to the skin a safe and effective amount of the composition of the present invention. The amount of composition applied, frequency of application and duration of use will depend on the level of the fig serum fraction and / or other components of a given composition and, for example, the age of the skin present in the subject. It varies greatly depending on the level of control and the level of control desired in light of the rate of further aging of the skin.

I. Physiological Activity of Fig Serous Fraction Also, the present invention provides for the pigmentation of the skin by topically applying to the hyperpigmented area a cosmetic composition for interfering with one or more steps in melanogenesis (melanin synthesis). It also relates to a method for reducing the appearance of excessive deposition. The cosmetic composition provides enzyme inhibition (trypsin inhibition activity and / or tyrosinase inhibition activity), antioxidant activity (ORAC and DPPH), and / or COX-2 inhibition, thereby causing one or more in melanogenesis. Interfere with the step.

  A method for preparing a botanical cosmetic composition exhibiting biological activity is currently available in that it provides a plant extract that captures the full spectrum of activity contained in plant cells. Conveniently over possible methods. As shown in Example 6, the fig extract of the present invention exhibits a higher physiological activity than the fig extracted by conventional means.

  In one embodiment of the present invention, a method of whitening mammalian skin comprises topically administering a cosmetic composition comprising an effective amount of a fig serum fraction to inhibit trypsin activity.

  Another embodiment of the invention includes a method of whitening mammalian skin by inhibiting tyrosinase activity in the skin of a mammal, the method comprising a cosmetic composition comprising an effective amount of fig serum fraction. Topically administering the product to the mammal.

  Normal skin color is formed by melanin, which is also a natural pigment that also determines body hair and eye color. In the skin, the enzyme tyrosinase is essential for the biochemical pathway involved in the conversion of the amino acid tyrosine to melanin. When too much melanin is produced and deposits are formed in the skin by these melanins, hyperpigmentation occurs. The cells that make up the pigment are called melanocytes. They are located at the base of the epidermis. Melanocytes produce melanosomes that pass over other cells of the epidermis and reach the upper layers of the skin. Melanin synthesis occurs only in melanosomes. If too much melanin is produced, deposits are formed and hyperpigmentation appears in the skin.

  Tyrosinase catalyzes o-hydroxylation from monophenol to the corresponding catechol (monophenolase or cresolase activity) and oxidation from monophenol to the corresponding o-quinone (diphenolase activity or catecholase activity) Monooxygenase. These functions of tyrosinase play an important role in the formation of melanin pigments during melanogenesis. Melanin production is primarily responsible for skin color and plays an important role in preventing sunlight-induced skin injury. However, abnormal accumulation of melanin products in the skin is responsible for hyperpigmentation, including melanosis, brown spots, freckles and senile moles, which results in an undesired aesthetic appearance There is a possibility (Jeon et al. (2005) Bull. Korean Chem. Soc, Vol. 26: 1135-1137).

  The present invention generally relates to the inhibition of the activity of at least one enzyme involved in skin pigmentation or coloration in mammalian skin tissue. Fig serous fraction can inhibit the activity of trypsin, as well as the activity of tyrosinase and other tyrosinase-like enzymes. Furthermore, the present invention also relates to anti-oxidant activities associated with dyes (ORAC and DPPH), including superoxide scavenging activity, and COX-2 inhibition.

  The cosmetic composition obtained from the serum has a superoxide scavenging potency that ranges from an ICR50 value of about 50-190 μg dry matter / ml. As used in this application, the term “ICR50 value” refers to the concentration of dry matter contained in the serous fraction of cells necessary to inhibit the reduction of cytochrome c by 50 percent.

  The compound may be administered to the mammal several times a day, or the compound may be administered less frequently, for example, once a day, once a week, once every two weeks, once a month. Or even less frequently, for example, once a few months, or administration may be no more than once a year. The frequency of administration will be readily apparent to those skilled in the art and will depend on any number of factors, including, but not limited to, the type and severity of the disease being treated, the type and age of the animal, and the like.

J. et al. Optional Components The compositions of the present invention may contain them, provided that the variety of other ingredients conventionally used in a given product type can tolerate changes that would benefit the present invention. it can. The composition can include a dermatologically acceptable carrier.

  The optional components, when incorporated into the composition, are, within reasonable judgment, free from undue toxicity, incompatibility, instability, allergic response, etc., and are intended for use in contact with human skin tissue. Should be suitable. The CTFA Cosmetic Ingredient Handbook, Second Edition (1992) describes a wide variety of non-limiting cosmetic and pharmaceutical ingredients commonly used in the skin care industry, which are Suitable for use in the composition. Examples of these component classes include abrasives, absorbents, aesthetic components, ie fragrances, pigments, colorants / colorants, essential oils, anti-hardening agents, anti-foaming agents, binders, biology Additive, buffering agent, bulking agent, chelating agent, chemical additive, colorant, cosmetic astringent, cosmetic biocide, denaturant, astringent as a drug, topical analgesic, film formation Agents or film materials, eg, polymers to aid film forming properties and dyeability of the composition (eg, copolymers of eicosene and vinyl pyrrolidone), opacifiers, pH adjusters, sprays, reducing agents, scavengers As well as thickeners.

  In some embodiments, it may be desirable to include a second, third, or fourth skin tone agent in combination with FSF in the composition. The second, third or fourth skin tone agent can be included to further improve the overall skin tone. The compositions of the present invention are preferably from about 0.1% to about 50%, more preferably from about 0.2% to about 20%, and even more preferably from about 1% to about 50% by weight of the composition. Contains 10% additional skin tone agent, if present. The amounts listed herein should only be used as a guide. This is because their potency varies considerably, so the optimal amount of additional skin tone agent depends on the particular active substance chosen. Preferred skin tone agents include, but are not limited to, N-acetylglucosamine, vitamin B3, and undecylenoylphenylalanine (eg, sold under the trade name Sepiwhite, Seppic, France). In some embodiments, one composition (eg, composition number 1 in Table No. 1) can be used as a local treatment for one or more hyperpigmentation spots, One or more other compositions (eg, Composition No. 2, No. 3, and No. 4 in Table No. 1) are more widely applied to the facial skin surface before or after specialized treatments to provide skin over the face The color tone can be improved.

  The topical compositions of the present invention are in a variety of forms, including but not limited to lotions, emulsions, mousses, serums, sprays, aerosols, foams, sticks, pencils, gels, creams and ointments. Can be provided. In one embodiment, the composition takes the form of a solution, and in another embodiment, the composition takes the form of a lotion.

K. Composition Preparation The compositions of the present invention are generally prepared by conventional methods, such as methods known in the art of making topical compositions. Such methods typically involve mixing the components in a relatively uniform state in one or more steps, which may or may not involve heating, cooling, applying a vacuum, etc. There may not be. The composition is preferably prepared to optimize the stability (physical stability, chemical stability, light stability) and / or delivery of the active material. This optimization is appropriate for pH (eg, less than 7), complexed with active agents, and thus the elimination of materials that may have undesirable effects on stability or delivery (eg, The elimination of contaminating iron), the use of approaches to prevent complex formation (eg packaging of suitable dispersing agents or double compartments), the use of suitable light stability approaches (eg tanning) Stop / sunblock integration, use of opaque packaging) and the like.

L. Method of treatment In one embodiment, the user selects a hyperpigmented spot for treatment and the first composition is applied to the hyperpigmented spot at least once a day, more preferably one day. Apply twice, at least about 4 weeks. In another embodiment, the first composition is applied to the selected hyperpigmented stain for at least about 8 weeks. The first composition can take any form. In one embodiment, the composition takes the form of a solution that is topically applied to a hyperpigmented stain using a dropper. Other applicators that can topically apply the first composition to the hyperpigmented stain may be used. For example, the composition may be pigmented using an applicator having a foam or cotton tip that releasably retains the first composition, such as a solution, lotion or other form described herein. Applicable to excess stains. In another embodiment, the composition is applied to one or more hyperpigmented stains and more generally simultaneously (ie within the same treatment cycle) by one or more facial skin surfaces.

  In some cases, the method of treatment comprises selecting a plurality of hyperpigmented spots for local treatment with the first composition during a single treatment cycle. As used herein, a treatment cycle refers to a single application of the composition to the intended skin surface. For example, a single application of a first composition to one or more hyperpigmented stains for a reasonably short period of time (eg, over a period of 1 to 30 minutes) may result in a single treatment cycle. Would constitute. In contrast, a single twice-daily application of the first composition to one or more hyperpigmented stains constitutes two treatment cycles, where the application is longer They are separated from each other (for example, there is an interval of 1-12 hours).

  In one embodiment, the method of treatment includes applying the first composition in combination with a second composition applied before or after the first composition, wherein the second composition comprises: Apply more generally to one or more facial skin surfaces to improve the overall tone appearance of the facial skin. The second composition can be applied to one or more of the frontal, perianal, chin, orbital, nasal and cheek skin surfaces. In one embodiment, the second composition is simultaneously applied to at least the cheek, forehead and chin / mouth circumference skin surface during a single treatment cycle. Compared to the local treatment of hyperpigmentation spots, a greater surface area is provided to which the second composition is applied.

  Some of the methods described herein contemplate applying the composition of the present invention using an applicator, but the applicator is not necessary and the composition of the present invention is applied directly. It will be appreciated that or may be applied using one finger (or in some other manner). Further, although one embodiment of the present invention contemplates topical application of the composition to hyperpigmented spots, the composition of the present invention is generally applied more than one or more facial skin surfaces. It will also be appreciated that the appearance of hyperpigmented stains lined up in those facial skin areas can be reduced.

  The following examples are provided to illustrate certain features and advantages of various embodiments of the invention, but the examples should not be construed as limiting their scope.

  The invention will now be described with reference to the following examples. These examples are provided for purposes of illustration only and should not be construed as limiting the present invention in any way whatsoever. It should be construed to include any modifications that become apparent as a result of the teaching provided in.

Preparation of a biologically active serous fraction obtained from fresh leaves of benghalensis (Ficus benghalensis).
FIG. 1 is a schematic diagram illustrating one embodiment of a process for preparing a biologically active serous fraction obtained from fresh fig leaves.

  A sufficient amount of fresh leaves of Ficus benghalensis was collected to obtain approximately 100 kg of dry matter. The level of dry matter in the fresh leaves was measured to be 32.01% and approximately 312.4 kg of fresh plant leaves were required to obtain 100 kg of dry matter. Care was taken to preserve the water content inherent in fresh leaves and avoid wilting due to water loss. Harvesting was performed to avoid or minimize any damage to the harvested fresh leaves. All steps were completed in the shortest possible period to minimize the exposure of fresh leaves to sunlight, high temperatures, and other undesirable environmental factors.

  The collected leaves were then washed at a water pressure of ≦ 1 kg / cm 2 for ≦ 5 minutes to remove soil particles and other debris from the leaves before further processing. The remaining wash water did not contain any green or brown pigment, indicating that the leaf tissue was complete, the water pressure and duration of wash was adequate. Excess water was removed from the washed leaves. The washed fresh fig leaf is then mechanically separated, thereby containing most of the intracellular material of parenchymal cells, free of fibers, cell juice and mainly containing cell walls, rich in fibers. The material was effectively separated. No exogenous solvent and water were added before or during the separation process.

  The washed leaves were crushed, macerated and pressed to obtain a liquid intracellular content (ie, cell juice), and the intracellular content was separated from the fiber rich material. Using a 5HP engine and a hammer mill with a series of screens (Model VS35, Vincent Corporation, Tampa, FL), the leaves were crushed to produce the appropriate small sized leaf tissue particles in the shortest possible time. The biomass temperature was obtained without significantly increasing. The hammer mill was set to produce maximum size macerated leaf particles ≦ 2.0 centimeters during the ≦ 10 second treatment. The temperature of the macerated fresh leaves only increased by ≦ 2 ° C.

  Cell juice was obtained from freshly macerated leaves using a horizontal continuous screw press (Compact Press CP-6, Vincent Corporation, Tampa, FL) equipped with a cone supported by compressed air. The pressure on the cone was maintained at a level of ≧ 15 g / cm 2 and a screw speed of 12 rpm was used. Under these conditions, the cell juice temperature only increased by ≦ 5 ° C.

  This treatment yielded a fiber-rich material and cell juice. Residual small fiber particles were further removed from the cell juice by purification using a continuous flow centrifuge (Model 12-413V, AML Industries, Inc., Hatboro, PA) with a fully automatic discharge unit. At a flow rate of 2 liters / minute, the retention time for cell juice purification at ≦ 2,250 g was ≧ 100 seconds. The above regimen produced cell juice containing no fiber. The precipitate containing small fiber particles was collected and combined with the remaining, fiber rich material that occurred after fresh leaf pressing.

  The above process allowed the production of 160.9 kg of cell juice with a dry substance content of 9.29% and 151.5 kg of fiber rich material with a dry substance content of 56.14%. . The cell juice was placed in a tightly closed 15 liter rectangular HDPE container without delay and frozen at −30 ° C. The solid state frozen cell juice was kept at this low temperature for further use.

  Cell juice is composed of three main types of components: (i) membrane-bound chloroplast, mitochondria, endoplasmic reticulum, nucleus, lysosome, peroxisome, vacuole, Golgi; and (ii) non-membrane bound Type ribosomes, microtubules; and (iii) components not related to the above groups, eg cytoplasm. Due to the presence of organelles and their fragments, and unwanted dyes and proteins in the cell juice, including but not limited to color, solubility, transparency, stability and in vitro activity, Fractionation was required to produce personal care ingredients with the desired combination of functional properties. To achieve these goals, the cell juice was fractionated using a variety of processes including cell juice fluidization, pH adjustment, focused microwave radiation, centrifugation and vacuum filtration. The isolated cell juice serum fraction was then stabilized with preservatives and antioxidants.

  The duration and intensity of cell juice treatment was minimized to eliminate oxidative stress, hydrolysis, denaturation, isomerization, polymerization and other unwanted processes.

  Conversion of the cell juice from the frozen state to the initial liquid state in a 15 liter container was achieved by fluidization over ≦ 2 minutes. During this treatment, the cell juice temperature only increased by ≦ 20 ° C. Due to the short duration of this treatment, it was possible to minimize both the denaturation process and oxidative damage. The physicochemical and biochemical properties of the cell juice after freezing and fluidization were identical to the corresponding properties measured during the separation of the cell juice from fresh leaves. Their properties included, but are not limited to, dry matter content, pH, conductivity, redox potential, osmotic pressure and IR spectrum.

  The pH of the cell juice near neutral was then adjusted using a titration method utilizing 5.0N hydrochloric acid (HCl) to reduce the cell juice pH to ≧ 3.0 (pH adjustment 1). . The conditioned cell juice was processed without delay by focused microwave radiation using a frequency of 2450 MHz. During this focused microwave treatment (FMP), the cell juice temperature increased temporarily to 90 ° C. and held at this temperature for 1 minute, and then the cell juice temperature immediately decreased to ≦ 30 ° C. The treated cell juice was then rapidly separated using a continuous flow centrifuge CEPA LE (Carl Padberg Zentrifugenbau GmbH, Germany) with a retention time of 15,000 rpm and ≧ 30 seconds. Separation of 15.0 kg of treated cell juice gives a bright brown color with a green colored paste-like precipitate (“Precipitate I”) 1.37 kg and a dry substance content of 6.75%, slightly 13.63 kg of a liquid supernatant emitting milky white light (“Supernatant I”) was obtained. This supernatant I was used for further fractionation.

  Table 1 shows the pH-regulated cell juice FMP for the dry matter content in supernatant I and its color, as well as the presence of chlorophyll a and chlorophyll b (determined by measurement of light absorption at 662 nm and 642 nm, respectively). Data relating to the effect of maximum temperature (Tmax) reached during processing is shown.

  The data in Table 1 shows that supernatant I obtained at Tmax = 90 ° C. has a higher dry matter content and no chlorophyll. For supernatant I obtained after Tmax = 60 ° C., the Gardner scale value is lower, but this preparation has a significantly lower dry matter content and a higher residual amount of chlorophyll. In addition to being an undesired presence in cosmetic ingredients, chlorophyll may be converted to pheophorbide, which is a toxic compound (Bergstrom, LC, Vucenik, I., Hagen, IK, Chernomorsky SA, Poretz RD In-vitro photocytotoxicity of lysosomotropic immunoliposomes containing pheophorbide a with human bladder carcinoma cells, J. Photochem. Photobiol., 24, 1, 17-23, 1994), considered to be involved in skin irritation (Kato T., Yamada K., Relationship between appearance of photosensitization and total pheophorbide level in spirulina powder, J. Food Hyg. Soc. Japan, 36, 632-634, 1995).

  Based on the above reasons, FMP Tmax = 90 ° C. of cell juice with adjusted pH was selected as the preferred type for obtaining supernatant I, and then using supernatant I, but not limited to these However, further fractionation was performed to improve functional properties, including color, transparency and stability of personal care ingredients. Supernatant I has desirable in vitro activities, such as (i) enzyme inhibitory activity, including but not limited to tyrosinase, elastase, trypsin, cyclooxygenase-2 (COX-2) inhibitory activity, (ii) It should be noted that it had antioxidant activity, including, but not limited to,) free radical scavenging activity, and (iii) oxygen radical absorption ability. Taking into account the critical importance of all of the above in vitro activities, their activity should not be affected by further processing necessary to improve the functional properties of the desired personal care ingredients. For composition improvement, the supernatant I should be further processed to significantly remove other undesired compounds, including brown pigments and residual proteins.

  To achieve this goal, supernatant I was subjected to further processing, including pH adjustment and separation. Initial treatment was triggered using a titration method utilizing 50% sodium hydroxide (NaOH) to increase the pH of cell juice supernatant I from about 3.0 to about 7.5 (pH adjustment 2 ). It should be noted that Supernatant II lost the desired elastase and trypsin inhibitory activity above pH = 7.5. As a result of pH adjustment 2, a darker material is produced and the opalescence is enhanced and this material is run on a continuous flow centrifuge CEPA LE (Carl Padberg Zentrifugenbau GmbH, Germany) with a retention time of 15,000 rpm and ≧ 30 seconds. Used and immediately purified. By the above separation, 0.53 kg of a brown colored paste-like precipitate (hereinafter, Precipitate II), and a supernatant having a dry substance content of 6.59%, colored brown and slightly emitting milky white light ( Hereafter, 13.10 kg of supernatant II) was obtained.

  Supernatant II was then subjected to titration utilizing 5.0N hydrochloric acid (HCl) to reduce the pH value to about pH 3.6 (pH adjustment 3). Such treatment resulted in a supernatant II that had a slight increase in opalescence but titrated a lighter pH. This material was processed using sterile filtration through a membrane having a pore size of 0.2 micrometers. This resulted in a bright, clear serum fraction of fresh fig leaves.

  The chromaticity value of the serum fraction (Gardner scale value = 7.0) was lower than the chromaticity value of supernatant I (Gardner scale value = 8.5). It should be noted that the Gardner scale chromaticity values of the serous fraction were always lower than the corresponding supernatant I chromaticity values obtained under different FMP Tmax conditions (Table 2).

  Serous fractions obtained from cell juices are fluidized, pH adjusted (within a pH range of 3.0-7.0), focused microwave radiation (Tmax = 90 ° C., 1 minute FMP), centrifugation and After sterile filtration, all desirable enzyme inhibitory activity, free radical scavenging activity and antioxidant activity were demonstrated.

  For the determination of residual protein content in serum fractions containing phenolic compounds that may interfere with colorimetric assays, the Kjeldahl method is used to determine the nitrogen content in the serum fraction and its ultrafiltrate. The amount was reliably detected. Using different membranes, the serum fraction was separated into three filtrates with molecular weights of ≦ 15, ≦ 10 and ≦ 5 kilodaltons (kD), respectively. Data on the nitrogen content in the sample is shown in Table 3.

  The data show that the nitrogen content did not change significantly even after ultrafiltration through membranes that fractionate low molecular weights, which in effect was found in the serum fraction. All of the nitrogens were non-protein, ie, the serum fraction shows no protein.

  Further stabilization of the serum fraction was achieved by adding antioxidants, stabilizers, chelating agents and preservatives. The following is the composition of the additive that was utilized to stabilize the serous fraction described in Example 1 presented: 0.2% sodium metabisulfite, 0.1% potassium sorbate, 0.1 % Sodium benzoate, 0.1% sodium methylparaben. The mixture was incubated until complete solubilization was reached (≧ 30 minutes). 1.9% pentylene glycol was then added to the mixture.

  The serum fraction contained approximately 6.38% dry matter, and its yield from fresh fig leaves was approximately 36%. The dry matter yield of the serous fraction from 100 kg dry matter of the first fresh fig leaf was approximately 7.2 kg.

  Selected characteristics of the serum fraction and its in vitro activity are shown in Tables 4 and 5.

Comparison of characteristics and in vitro activity of serous fractions obtained from cell juice of Ficus benghalensis Fresh fig leaves were collected at different locations and processed as described in Example 1 to produce cell juice. Obtained. The cell juice was frozen and stored at −30 ° C. in a tightly closed 15 liter rectangular HDPE container. Using the same procedure as described in Example 1, one or more containers were processed at one time to obtain a serum fraction.

  The data shown in Table 6 and Table 7 are obtained from serum fractions obtained from multiple fractions of cell juice frozen at different times of the same source and from different sources of frozen cell juice. 2 shows selected characteristics of the serum fraction and variability of activity in vitro.

Preparation of water extract of dried leaf of bengal tiger (Ficus benghalensis) 50 g of air-dried leaf of tiger beetle (Ficus benghalensis) (collected from the same batch of leaves used in Example 1) was collected from GM200 Grindomix knife mill (Retsch, Germany). ) To obtain particles having a size of <300 micrometers. Milling included 2500 rpm for 20 seconds followed by 2500 rpm for 10 seconds and then 10,000 rpm for 10 seconds. The ground leaves were homogenized with deionized water using an OMNI Programmable Digital Homogenizer (OMNI International, Kennesaw, GA). 35 g of ground leaves were mixed with 490 g of water and placed in an ice bath on the homogenizer platform. Homogenization was performed at 15,000 rpm for 15 minutes using a 20 mm homogenizer generator. The homogenate was then subjected to microwave treatment for 1 minute at 90 ° C. in the Initiator 2 Focused Microwave Processor (Biotage AB, Uppsala, Sweden). The microwaved material was then centrifuged at 3,200 g for 30 minutes. The supernatant was then filtered under vacuum through three layers of Whatman # 2 paper and the pH of the filtrate was titrated to pH 4.0 using hydrochloric acid (HCl). The pH titrated material was centrifuged at 3,200 g for 30 minutes, and then the supernatant was filtered through a 0.2 micrometer sterile filter under vacuum. Stabilizers, ie 0.2% sodium metabisulfite, 0.1% potassium sorbate, 0.1% citric acid, 0.1% sodium benzoate were added to the sample. The mixture was incubated until complete solubilization was reached (≧ 30 minutes). The resulting dried leaf water extract was placed in a glass vial and stored at room temperature in the dark. Selected characteristics and in vitro activity of the water extract of dried fig leaves are shown in Table 8.

  Within a wide range of concentrations tested, the dried fig leaf aqueous extract showed no inhibitory activity of elastase, trypsin and cyclooxygenase-2. A comparison of the water extract and serous fraction obtained from the same batch of Ficus benghalensis leaves for selected characteristics and in vitro activity is shown in Table 9.

Characteristics and in vitro activity of serous fractions from different Ficus species and locations In addition to fresh leaves of Ficus benghalensis collected in India and Florida (USA), fresh leaves of the following Ficus species Serum fractions were obtained using for fractionation: Ficus carica, Ficus elastica, Ficus microcarpa, and Ficus trigonata. With the exception of Ficus trigonata grown in Puerto Rico, these Ficus species were grown in Florida, USA.

  Serous fractions were obtained by the procedure described in Example 1. All these fractions were compared with respect to their yield, selected physicochemical properties and in vitro activity (Table 10, Table 11 and Table 12).

  The above data show that among different Ficus species, the dry matter content in fresh leaves, the yield of cell juice and the serum fraction, and their dry matter content, color and pH are very prominent. It shows that it fluctuated. Corresponding differences between two Ficus benghalensis grown in India and Florida appeared lower than differences between different Ficus species.

  This conclusion is supported by additional data regarding comparison of selected physicochemical characteristics (Table 11) and in vitro activities (Table 12) of serous fractions obtained from different Ficus species.

Comparison of LC / UV / MS chromatograms of traditional fig extract (ficus benghalensis) and fig serum fraction, after UV separation on C18 column, 240-500 nm UV detection and cation Fig extract and FSF components were detected by both electrospray mass spectrometry in both (m / z 150-1150) and anion (m / z 100-1100) modes. Due to the fast scan speed utilized on the quadrupole MS, only major components and / or components with high ionization efficiency were observed in the mass chromatogram. The fig extract from the fig serum fraction was analyzed by TOF / MS and the structural assignments are based on this accurate mass and in-source fragmentation data.

  As shown in FIG. 2, conventional fig extracts contain slower eluting (more hydrophobic) compounds that are not detected in FSF. As shown in FIG. 3, one group of slow eluting compounds appears to be pheophorbide, which are chlorophyll degradation products. FIG. 4 shows that the conventional extract contains higher amounts of components that appear to be flavonol glycosides. As shown in FIG. 5, FSF has higher levels (about 10 ×) of catechins and associated condensed tannins. However, as shown in FIG. 6, the levels of the three caffeoylquinic acid isomers did not appear to differ substantially between the two samples. FIG. 7 shows that FSF contained higher levels of free tyrosine, phenylalanine and tryptophan.

Method:
Fig Serous Fraction Sample Preparation:
FSF was prepared as in Example 1. FSF samples were diluted 50-fold with 90:10 water: DMSO (20 μL sample + 100 μL DMSO + 880 μL water) and analyzed by LC / UV / MS according to the following conditions. The approximate solids content in the final sample was about 1.26 mg / mL.

Conventional sample preparation:
10.64 mg of sample was weighed into a 4 ml glass vial. 1.064 mL DMSO was added to the vial, sonicated for 30 minutes, and vortexed occasionally to mix. 100 μL of this sample was added to a 4 mL glass vial and diluted with 900 μL of water. Approximate solids content in the final sample about 1 mg / mL.
HPLC conditions:
HPLC: Waters Acquity UPLC Binary Solvent Manager: S / N M05UPB601M
Waters Acquity UPLC Sample Manager: S / N M05UPS632M
Waters Acquity UPLC PDA Detector: S / N M05UPD879N
MS: Waters Micromass Quattro Premier MS: S / N VAA-219
LC column: Waters Acquity UPLC BEH C18, 1.7 mm, 2.1 × 100 mm, part number 186002352, lot number 0150371186
Mobile phase: A: Water with 0.1% formic acid B: Acetonitrile with 0.1% formic acid Separation: Gradient (see table)

Injection volume: 7.5μL, partial loop with needle overfill
Column temperature = 25 ° C
PDA: 240-500 nm, 20 points / second, filtration time: constant at 0.2 seconds, exposure time = automatic, resolution: 1.2 nm
MS conditions:

Melanin synthesis B16-F1 mouse melanoma cell line is utilized in the assay. B16-F1 cells are obtained from the American Tissue Culture Collection, Virginia, USA. The cell culture medium used in the assay includes 500 mL Dulbecco's Modified Eagle Medium (DMEM), 50 mL fetal bovine serum (FBS), and 5 mL penicillin-streptomycin solution. B16-F1 cells grown in this medium and grown to a culture density greater than 90% synthesize melanin. While not intending to be bound by any theory, it is postulated that melanin synthesis is stimulated by stress induced by media and / or growing to high culture densities. DMEM and FBS can be obtained from the American Tissue Culture Collection, and penicillin-streptomycin solution can be obtained from Invitrogen, Inc. , California, USA. The instrument used in the assay is a CO2 incubator, such as Forma Series Model 3110, Therma Scientific, Massachusetts, USA; a hemocytometer, such as the Bright Line model, Hausen Scientific, Hauser Scientific, -Visible spectrum plate readers, including, for example, SpectraMax 250, Molecular Devices, California, USA. The assay steps include:

  Day 0-Cell growth: Warm the cell culture medium to 37 ° C and place 29 mL in a T-150 flask. Approximately 1 × 10 6 B16-F1 passage 1 mouse cells are added to a T-150 flask and incubated for 3 days at 37 ° C., 5% CO 2, 90% relative humidity to a culture density of about 80%. To do.

  Day 3—Start 96-well plate: On day 3, the cells obtained from the T-150 flask are trypsinized and the cell concentration determined using a hemocytometer. A 96-well plate is started with 2,500 cells per well in 100 μL of cell culture medium. Plates are incubated for 2 days at 37 ° C., 5% CO 2, 90% relative humidity to a culture density of at least 20% to 40%.

  Day 5—Remove cell culture medium from plate and replace with fresh medium (100 μL per well). [Water or DSMO] 1 μL of [test compound] diluted in solvent is added. Multiple dilution ratios can be tested to generate a dose response curve, preferably three wells are treated with each dilution ratio. Controls include wells with cell culture medium, B16-F1 cells and solvent (control number 1); wells with cell culture medium and solvent (control number 2); and, optionally, background of [test compound] If control over color is required, a well containing cell culture medium, solvent and [test compound] (control number 3) is included.

  Day 7-Measurement of melanin production: Cells should have a culture density greater than about 90%. Otherwise, this data point is not used. 100 μL of 0.75% sodium hydroxide solution is added to each well. Using a UV-Vis plate reader at 410 nm, a 96-well plate is read to optically measure the amount of melanin produced between wells treated with [test compound] and untreated control wells. Measure automatically. Wells in which melanin is produced appear brown. Wells with little melanin production appear clear to bright purple. The percent inhibition of melanin synthesis is calculated by the following formula:

In the formula, OD410 is an optical density at 410 nm as measured by UV-Vis Spectrum Plate Reader.

  When using control number 3, the formula for inhibition of percent melanin synthesis is

It is.

  In general, using the assay outlined above, melanin synthesis was inhibited in FSF treated B16-F1 cells compared to control cells, as shown in Table 13 below.

  Considering variables such as the complexity of melanin production and movement within the skin, and the ability of test compounds to penetrate the skin, this assay allows for in vivo outcomes for facial hyperpigmentation spots in humans. Although not necessarily asserted, this assay shows that materials such as FSF can potentially affect tyrosinase activity.

Inhibition of tyrosinase Tyrosinase is an important enzyme in the biosynthesis of melanin. This assay can identify agents that can interfere with the ability of the mushroom tyrosinase enzyme to convert L-tyrosine to L-dihydroxyphenylalanine (L-DOPA).
Reagents and supply tyrosinase enzyme: mushroom tyrosinase, available from Sigma-Aldrich, Missouri, USA,
Enzyme substrate: L-tyronsine, available from Sigma-Aldrich, Missouri, USA,
Buffer: phosphate buffered saline (PBS), available from Invitrogen, California, USA,
Positive control: 4-hydroxyphenyl-β-D-glucopyranoside (arbutin), available from Sigma-Aldrich, Missouri, USA,
Dimethyl sulfoxide (DSMO), available from Sigma-Aldrich, Missouri, USA
Falcon® 1172 Microtestt non-tissue culture treatment, clear flat bottom 96 well plate,
A potential tyrosinase inhibitor,
Well plate reader: Spectra MAX Plus, available from Molecular Devices, California, USA,
Data acquisition and analysis software: Available from SoftMax Pro, Molecular Devices, California, USA.
Working solution concentration Final concentration tyrosinase enzyme in assay: 26 units / mL 13 units / mL
L-tyrosine substrate: 1 mM 0.5 mM
Arbutin positive control: 20 mM 200 uM

Assay protocol:
Preparation of Reagents and Positive Control A 1 mM enzyme substrate working solution is prepared by adding 0.01812 g L-tyrosine to 100 mL 1 × PBS. Sonicate until L-tyrosine is dissolved. Vortex as necessary. Store at 4 ° C when not in use.

  Arbutin positive control 0.2 M stock solution is prepared by adding 0.0544 g arbutin to 1 mL DMSO. Vortex and sonicate for 1 minute until arbutin is dissolved. This solution is diluted 1:10 by adding 100 μL to 900 μL DMSO to give a 20 mM arbutin working solution. Store at room temperature until use.

  Potential tyrosinase inhibitors should be prepared in DMSO. The final volume of test compound in the assay is 2 μl, so the working solution is typically composed of 5-40 mM (100 ×), which gives a final concentration of 50-400 μM in the assay.

  Reconstitute tyrosinase enzyme at 1000 U / mL using cold 1 × PBS. This stock solution is stored at -20 ° C in the dark in 1 mL aliquots until needed. Prepare a 26 U / mL enzyme working solution by adding 1 ml of thawing stock solution (1000 U / mL) to 37.5 cold 1 × PBS buffer. This is sufficient to handle four 96 well plates. Keep out of light and on ice until used in the assay.

Performing the assay 200 μL of 1 × PBS buffer is added to triplicate wells on each test plate to obtain an appropriate blank.

  2 μL of DMSO is added to triplicate wells to obtain a vehicle control.

  2 μL arbutin is added to triplicate wells to obtain a positive control.

  2 μL of potential tyrosinase inhibitor is added to triplicate wells.

  Remove the blank and add 98 μL tyrosinase enzyme working solution to each well. Compound and enzyme are mixed by pipetting up and down twice or vortexing briefly.

  Add 100 μL / well of L-tyrosine substrate.

  Select the setting for kinetic measurements on a SpectraMax 250 plate reader and record the absorbance reading at 475 nm for 1 hour every minute.

  Using data acquisition software, slopes are calculated for control and test compounds.

  The percent tyrosinase inhibition is calculated by the following formula:

  In general, using the assay outlined above, FSF inhibited tyrosinase activity, as shown in Table 14 below.

  Considering variables such as the complexity of melanin production and movement within the skin, and the ability of test compounds to penetrate the skin, this assay allows for in vivo outcomes for facial hyperpigmentation spots in humans. Although not necessarily asserted, this assay shows that materials such as FSF can potentially affect tyrosinase activity.

In vivo study on reduction of hyperpigmentation spots and melanin uniformity A 9 week in-vivo study included a 1 week normalization period using round robin, vehicle control, facial segmentation design The experiment was conducted using 270 subjects. 270 subjects were screened according to inclusion / exclusion criteria including:
Inclusion—has hyperpigmented spots on both sides of the face, around the cheeks and / or around the orbit.
-At least one 8-10 mm diameter hyperpigmented spot, 4 4-6 mm diameter spots, or 10 2-3 mm diameter spots (sunlight) in the cheek area on each side of the face Stains, freckles or melanosis stains) or equivalent stain areas.
-Willing to refrain from sun exposure by using physical UV blockers such as supplied UV lotions and hats to avoid facial sunburn, sunburn or windburn.
Exclusion-has been diagnosed as having atopy, eczema, psoriasis or other chronic skin disease.
-With obvious signs of facial skin disease (eg more than 5 comedones, areas of red scaled skin, superficial thin blood vessels, etc.).
-Has a noticeable area of discoloration or scarring on the face.
-Has more than 3 prominent nevi (<3 mm) on the face.

  270 subjects were mobilized for the study. Approximately 60 subjects dropped out during the course of the study. Each subject received two coded test formulations and applied to each half of the face twice a day. Facial treatment site images were acquired after baseline (week 0) and after 4 and 8 weeks of treatment and analyzed for changes in skin color and stain size and color. The product formulation included vehicle control, vehicle + 0.55% FSF, and vehicle + 5% vitamin B3 compound (niacinamide).

  Non-contact spectroscopic intradermal analysis (SiaCopy, Astron Clinica, UK) was used to collect and analyze images of the subject. This method used a digital camera (eg, Fuji S2 digital SLR) as a brand spectrometer and an instantaneous illumination source (eg, Sigma Super flash light source) to collect facial chromophore information. A cross polarizing filter was placed in front of the camera and illumination source to eliminate specular reflection.

  The chromophore mapping of the concentration and distribution of eumelanin (melanin) and oxyhemoglobin produces a grayscale concentration map for each of these chromophores. Image analysis software such as Optimas 6.5 can be used to select the band of interest in each chromophore map, from which the average grayscale value and spot area portion are calculated. The stain area portion indicates the total area occupied by the melanin stain as a percentage of the entire target band. A description of one type of chromophore mapping can be found in EP 1,810,614 and “The Distribution of Melanin in Skin Determined In Vivo”, British Journal of Dermatology, 2007, pages 620-628.

  FSF gave the best results after 4 weeks and significantly (p <= 0.10) reduced hyperpigmentation spots than the control and 5% vitamin B3 compositions. After 8 weeks, the vitamin B3 composition gave the best results, but the FSF composition was also significantly better than the control in reducing hyperpigmentation spots. Table 16 summarizes the image analysis data, where SAF is the average spot area and ΔSAF is the average change in the spot area from the baseline (week 0).

  The FSF was significantly better than the vehicle for SAF and directional for melanin uniformity better than the vehicle.

Analytical Methods The following analytical methods are used to determine various physical and chemical properties reported in the examples.

Method for determination of dry matter The dry matter level (percentage) was determined by comparing the weight of the liquid sample with the weight of the dry residue after evaporation of the liquid components. Disposable aluminum weighing pan, Ohaus Explorer E00640 balance, manufactured by Ohaus Corporation (Pine Brook, New Jersey), and Shel Lab model 1400E oven, manufactured by VWR (West Chester, Pennsylvania). The sample was dried in an oven set at 105C for 12 hours. The “wet” weight was obtained by subtracting the weight of the tare from the weight of the tare containing the liquid sample. The weight of the tare containing the same sample after drying was subtracted from the weight of the tare to obtain a “dry” weight. The level of dry substance is then equal to the “dry” weight divided by the “wet” weight and multiplied by 100%.

Method for Color Determination Using the Lovibond Comparator 3000 (Tinometer Limited of Salisbury, UK) device, following the standard procedure for the device according to the instruction manual, the color of the test article in the clear glass tube and the device The color (Gardner scale, 0-18) was determined by comparing the colored glass standard set in the two rings.

Method for determination of osmotic pressure The osmotic pressure was determined by measuring the freezing point depression of the solution and comparing it with the freezing point of the pure solvent. This measurement was performed using the Advanced Model 3250 Single-Sample Osmometer, Advanced Instruments, Inc. (Norwood, MA) and performed according to standard procedures for devices according to the instruction manual.

Method for Refractive Index Determination The refractive index is determined according to the standard procedure for the device according to the instruction manual on an Arias 500 refractometer, connected to an external temperature controlled circulator, manufactured by Reichert Analytical Instruments (Depew, NY). It was measured.

Method for measurement of parameters of the UV spectrum An Ultraspec 4300pro UV / visible spectrophotometer, Biochrom Ltd., which has a cell holder with a fluid jacket and leads to an external temperature-controlled circulator. (Cambridge, UK) determined the peaks, valleys and bends in the UV absorption spectrum. A quartz cuvette with an optical path length of 1 cm was used for samples diluted with deionized water. SWIFT II software package software, Biochrom Ltd. Device control and data analysis were performed from the Wavescan application manufactured by the manufacturer.

Method for Determination of Elastase Inhibitory Activity 96 well microtiter plates (Corning 3641), Corning Incorporated (Corning, NY) and Synergy 2 microplate reader, BioTek Instruments, Inc. Elastase inhibitory activity was determined by a colorimetric assay that measures the reaction rate configured for use with (Winooski, VT). Enzymatic activity to cleave the substrate was indicated by the occurrence of yellow color measured as an increase in absorbance at a wavelength of 410 nm. N-methoxysuccinyl-Ala-Ala-Pro-Val-pNA substrate (EPC FH237) and elastase (EPC SE563) were obtained from EPC (Elastin Products Company, Inc., Owensville, MO). The reaction volume in each well was 200 microliters, the elastase concentration was equal to 0.87 units / ml and the substrate concentration was equal to 363 μM. This procedure is described in Elastin Products Company, Inc. The method of the title “Assay with N-MeO-Suc-Ala-Ala-Pro-Val-pNA (EPC No. FH237) as substrate” from page 84 of Research Biochemicals Catalog (2004, p. 92) was revised.

Method for Determination of Cyclooxygenase-2 Inhibitory Activity Cyclooxygenase-2 (COX-2) inhibitory activity was determined by Cayman Chemicals COX inhibitor screening ELISA assay kit 560131.

Method for determination of antioxidant activity Synergy 2 microplate reader BioTek Instruments, Inc. (Performed Oxygen Radical Absorbance Capacity (ORAC) Assays with Biotech, available from (www.biotek.com/resources/docs/ORAC_Assay_Application_Note.pdf) for use with (Winooski, VT). Antioxidant activity was determined by ORAC test using a modification of the method described in the application note “Synergy HT Multi-Detection Microplate Reader”. In this assay, AAPH (2,2′-azobis-2-amino-propane) generates reactive oxygen species that damage the fluorescent probe (sodium fluorescein). Antioxidants such as (R) -Trolox methyl ether prevent or slow this damage and their effects can be quantified by fluorescence measurements. Using the excitation wavelength set at 485 nm and the emission wavelength set at 528 nm, a fluorescence reading was obtained, the reaction volume was 200 microliters, the AAPH concentration was 55 mM, the sodium fluorescein concentration was 1.33 μM, ( The R) -trolox methyl ether concentration range was between 80 μM and 2 μM. Fluorescein sodium (Fluka 46960), AAPH (Sigma 440914), and (R) -trolox methyl ether (Fluka 93509) were obtained from Sigma-Aldrich (St. Louis, MO). The AUC (area under the curve) value was calculated as the sum of the ratios (current fluorescence reading for the well divided by the first fluorescence reading for the well). The average of the AUC values for wells with deionized water was subtracted from the AUC for wells with (R) -Trolox methyl ether and wells with test articles to obtain an AUC corresponding to the storage of fluorescence by antioxidants. . A calibration curve was generated as a function of AUC associated with well antioxidants to show ORAC activity corresponding to (R) -trolox methyl ether weight. The ORAC activity for the test article was then calculated as the unit weight test article required to achieve an antioxidant effect equal to 1 provided by one unit weight of (R) -Trolox methyl ether.

Method for determination of capture activity Glass-coated polypropylene 96 well microtiter plate (Cat. No. 400062), SUN-SRi (Rockwood, TN) and Synergy 2 microplate reader, BioTek Instruments, Inc. A free-radical scavenging activity, ie DPPH (2,2-diphenyl-1-picrylhydrazyl) free, by a colorimetric assay measuring the reaction rate, adapted for use with (Winooski, VT) The radical scavenging activity was determined. Absorbance was measured at a wavelength of 515 nm. The reaction volume in each microplate well was 200 microliters and the initial concentration of DPPH was equal to 114 μM. L-ascorbic acid was used as a positive control. DPPH (Sigma D9132) and USP L-ascorbic acid (Sigma A-2218) were obtained from Sigma-Aldrich (St. Louis, MO). The stoichiometry of the reaction was calculated and expressed as the unit weight test article required to quench one unit weight of DPPH. This method is described in the article `` Use of a free radical method to evaluate antioxidant activity '' published by LWT-Food Science and Technology, Volume 28, Issue 1, 1995, pages 25-30, by W. Brand-Williams et al. Adapted from the procedure described in.

Procedure for rapid stability test using temperature profiling The procedure for rapid stability test using temperature profiling is shown below:
1. Operate 12 heating blocks, heat at specified temperature, operate refrigerator and maintain at specified temperature for at least 4 hours before experiment. In addition to the twelve samples placed in the heating block, one sample is placed at room temperature and another sample is refrigerated to obtain a total of 14 different temperatures. The temperatures are 5, room temperature (about 23C), 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72 and 75 ° C. The heating block is equipped with a block of aluminum inserts rolled to fit a 20 ml vial. These insert blocks fit inside the heated pocket block of the heating block. Each heating block should have an independent temperature controller and temperature sensor, and the temperature of the reaction block should be controlled at 0.1C.
2. Fill 14 vials with as little headspace as possible. Vial sizes may range from autosampler vials to scintillation vials as long as they are all the same size. For the purposes of this example, a 20 ml vial was used.
3. A label with a notebook reference and temperature is prepared for each vial. It is best to make the label as small and narrow as possible. If a color photograph is used to evaluate the color, one separate label without temperature is made and a vial is presented.
4). Each label is attached to the corresponding vial without obstructing the view of the product in the vial. This is usually done by sticking the label to the lid with a long vertical tab that reads from top to bottom. Place the label on the side of the lid, opposite the side with some markings on the vial, and therefore when taking a picture, display the label so that the sample in the vial is best viewed it can.
5. When assessing color, an initial photograph of the sample is taken using a digital camera. Allow the sample in the vial to be clearly seen (change the direction of the vial so that neither the label nor the print on the vial obstructs the view), and the sample is lowest from left to right Line up from temperature to highest temperature. A separate label is presented so that the separate label can be easily read in the image. To achieve consistency, the vial and camera positions can be marked on the laboratory bench, so this arrangement can be reproduced later. It is recommended to turn off the flash.
6). Photographs are taken of these types of samples as follows.
a. Clear solution: Use a flash against a white background and do not use a table lamp.
b. Opaque sample: turn off flash against black background, use table lamp When performing a chemical analysis, a sample is taken from each vial to obtain an initial reading.
8). Samples are placed in a heating block and refrigerator and the day and time are recorded.
9. At selected time points (by default, use days 3, 7 and 14), remove samples from heating block and refrigerator and allow to return to room temperature over at least 30 minutes.
10. If the color is to be evaluated, a new photo is taken using the vial and the camera location mark made in step 4. This is repeated at each time point.
11. If chemical analysis is to be performed, then each vial is sampled at this point. Repeat for each time point.
12 To assess color, pick the time points that best identify the different products and crops, label each product, and paste the photos together. A line depicting the time corresponding to 6 months and 1 year of room temperature can be drawn on the image using the stability table. At a given time point, find the accelerated temperature corresponding to 6 months and 2 years on the table, and this will determine which vial should be drawn between. (See, for example, FIG. 10)
13. For chemical analysis, the concentration is plotted versus temperature for each time point. Draw a smooth line through the data and record the temperature at which an unacceptable threshold is reached. In the stability table, this temperature is referred to time, and the time corresponding to room temperature is obtained. This technique can be applied to color analysis when using a Lab chromaticity meter to measure color and plot the color difference instead of density.
14 The stability table assumes an activation energy of 25 kcal / mol. Most hydrolysis and similar reactions are about this energy or higher. At higher energies, the product is more stable and the room temperature stability predicted by the table is lower than the actual stability (a very conservative estimate). Sometimes the energy is lower. For this reason, if a chemical analysis is performed, it is recommended to take the data generated according to the above and calculate the reaction energy using the Arrhenius plot to confirm that the assumption is correct.
15. Appendix: Lab color extraction from photos:
i. Move all photos onto the CD. The average Lab color of each sample is measured using color measurement software, for example, Optimas, and a computer equipped with any necessary peripherals.
ii. Start with the leftmost 5C sample, move from the upper right, and drag to the lower left to select the area where the vial colors are averaged. A 5C sample is set as a standard reference (this is only done the first time using a 5C sample).
iii. Record the L, a, b, Std Dev, and dEcmc values for each temperature sample. For each photo, the white point is recorded (should be 255, 255, 255).

  It should not be understood that the dimensions and values disclosed herein are strictly limited to the exact numerical values recited. Instead, unless otherwise stated, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as “40 mm” is intended to mean “about 40 mm”.

  All references cited in the “DETAILED DESCRIPTION” of the present invention are hereby incorporated by reference in their entirety. Citation of any description shall not be construed as an admission that it is prior art with respect to the present invention. To the extent that the meaning or definition of any term in the present description contradicts the meaning or definition of any of the same terms in the description incorporated by reference, the meaning or The definition shall prevail.

  While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. Let's go. Accordingly, the appended claims are intended to cover all such changes and modifications as fall within the scope of the invention.

Claims (34)

  A composition obtained from fresh fig leaf juice, which is a fig serum fraction. The fig cell serum fraction is
a. Separating the fig cell juice from a clean, fresh, unwielded fig leaf to obtain a fresh fig cell juice, wherein no exogenous liquid is added before or during said separation; and
b. Filtering the fresh fig cell juice to obtain a fiber-free cell juice;
c. Fractionating the cell-free cell juice to obtain a fig serum fraction, the fraction comprising:
(1) removing chlorophyll from the cell juice not containing the fiber to obtain supernatant I;
(2) removing pigment and protein from supernatant I to form a fig serum fraction, and (3) optionally adding a stabilizer to said fig serum fraction. The composition of claim 1 produced by a process comprising. Said removal of dye and protein from supernatant I,
i. Adjusting the pH of supernatant I to about 7.5 to form pH-adjusted supernatant I;
ii. separating pH adjusted supernatant I into precipitate II and supernatant II;
iii. Adjusting the pH of supernatant II to about 3.6 to form pH-adjusted supernatant II,
iv. 3. The composition of claim 2, comprising separating the pH adjusted supernatant II into a precipitate III and a fig serum fraction.   4. The composition of claim 3, wherein the stabilizer is selected from the group consisting of antioxidants, chelating agents, preservatives, and mixtures thereof.   5. The composition of claim 4, wherein the stabilizer is selected from the group consisting of sodium metabisulfite, potassium sorbate, sodium benzoate, methyl paraben sodium, pentylene glycol, and mixtures thereof.   4. The composition of claim 3, which is stable for at least 6 months at room temperature.   7. A composition according to claim 6, which is stable for at least 12 months at room temperature.   8. The composition of claim 7, which is stable at room temperature for at least 24 months.   The fig leaf is from a Bengal sage (F. benghalensis), a fig (F. carica), a ficus elastica, a banyan (F. microcarpa), a ficus trigonata, and combinations thereof. 4. The composition of claim 3, wherein said composition is selected from the group of Ficus species.   10. The composition of claim 9, wherein the fig serum fraction is substantially free of pheophorbide.   4. The composition of claim 3, wherein the fig serum fraction is substantially free of protein as measured by the Kjeldahl method.   4. The composition of claim 3, wherein the fig serum fraction is water soluble.   4. The composition of claim 3, wherein the fig serum fraction has a Gardner chromaticity value of less than 8.   14. The composition of claim 13, wherein the Gardner chromaticity value is less than 7.5.   The composition of claim 1, wherein the fig serum fraction has a biological activity capable of reducing hyperpigmentation of the skin.   16. The composition of claim 15, wherein the biological activity is sufficient to interfere with one or more steps in melanogenesis (melanin synthesis).   The composition of claim 16, wherein the biological activity is sufficient to inhibit melanogenic enzyme activity.   18. The composition of claim 17, wherein the enzyme activity is trypsin activity, tyrosinase activity, or both.   17. A composition according to claim 16, wherein the biological activity is sufficient to inhibit elastase activity.   The composition of claim 16, wherein the biological activity is sufficient to inhibit COX-2 activity.   17. The composition of claim 16, wherein the biological activity is sufficient to increase antioxidant activity, free radical scavenging activity, or both. A process for making a fig cell serous fraction composition comprising:
a. Separating fig cell juice from clean, fresh, unwielded fig leaves to obtain fresh fig cell juice, wherein no exogenous liquid is added before or during said separation;
b. Filtering the fresh fig cell juice to obtain a fiber-free cell juice;
c. Fractionating the cell-free cell juice to obtain a fig serum fraction, the fraction comprising:
(1) removing chlorophyll from the cell juice not containing the fiber to obtain supernatant I;
(2) removing pigment and protein from supernatant I to form a fig serum fraction, and (3) optionally adding a stabilizer to said fig serum fraction. Including processes. Said removal of dye and protein from supernatant I,
i. Adjusting the pH of supernatant I to about 7.5 to form pH-adjusted supernatant I;
ii. separating pH adjusted supernatant I into precipitate II and supernatant II;
iii. Adjusting the pH of supernatant II to about 3.6 to form pH-adjusted supernatant II,
iv. 23. The process of claim 22, comprising separating the pH adjusted supernatant II into a precipitate III and a fig serum fraction.   24. The process of claim 23, wherein the stabilizer is selected from the group consisting of antioxidants, chelating agents, preservatives, and mixtures thereof.   25. The process of claim 24, wherein the stabilizer is selected from the group consisting of sodium metabisulfite, potassium sorbate, sodium benzoate, methyl paraben sodium, pentylene glycol, and mixtures thereof.   26. The process of claim 25, wherein the composition is stable for at least 6 months at room temperature.   27. The process of claim 26, wherein the composition is stable for at least 12 months at room temperature.   28. The process of claim 27, wherein the composition is stable for at least 24 months at room temperature.   The fig leaf is from a Bengal sage (F. benghalensis), a fig (F. carica), a ficus elastica, a banyan (F. microcarpa), a ficus trigonata, and combinations thereof. 23. The process of claim 22, wherein the process is selected from the group of Ficus species.   23. The process of claim 22, wherein the composition is substantially free of pheophorbide.   23. The process of claim 22, wherein the composition is substantially free of protein as measured by the Kjeldahl method.   23. The process of claim 22, wherein the composition is water soluble.   23. The process of claim 22, wherein the composition has a Gardner chromaticity value of less than 8.   23. The process of claim 22, wherein the composition has a Gardner chromaticity value of less than 7.5.